EP4007813A1 - Rna-moleküle zur modulation der blüte bei pflanzen - Google Patents

Rna-moleküle zur modulation der blüte bei pflanzen

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Publication number
EP4007813A1
EP4007813A1 EP20850405.0A EP20850405A EP4007813A1 EP 4007813 A1 EP4007813 A1 EP 4007813A1 EP 20850405 A EP20850405 A EP 20850405A EP 4007813 A1 EP4007813 A1 EP 4007813A1
Authority
EP
European Patent Office
Prior art keywords
sequence
rna molecule
ribonucleotide
rna
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP20850405.0A
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English (en)
French (fr)
Other versions
EP4007813A4 (de
Inventor
Jonathan Paul ANDERSON
Ming Bo Wang
Neil Andrew Smith
Christopher Andrew Helliwell
Stephen Mark SWAIN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Commonwealth Scientific and Industrial Research Organization CSIRO
Original Assignee
Commonwealth Scientific and Industrial Research Organization CSIRO
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/AU2019/050814 external-priority patent/WO2020024019A1/en
Priority claimed from AU2020900327A external-priority patent/AU2020900327A0/en
Application filed by Commonwealth Scientific and Industrial Research Organization CSIRO filed Critical Commonwealth Scientific and Industrial Research Organization CSIRO
Publication of EP4007813A1 publication Critical patent/EP4007813A1/de
Publication of EP4007813A4 publication Critical patent/EP4007813A4/de
Pending legal-status Critical Current

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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Definitions

  • the present invention relates to new double stranded RNA (dsRNA) structures and their use in modulating flowering in plants.
  • the present invention also relates to methods of modulating the time of plant flowering.
  • RNA silencing is an evolutionarily conserved gene silencing mechanism in eukaryotes that is induced by double-stranded RNA (dsRNA) which may be of a form designated hairpin structured RNA (hpRNA).
  • dsRNA double-stranded RNA
  • hpRNA hairpin structured RNA
  • Dicer proteins In the basic RNA silencing pathway, dsRNA is processed by Dicer proteins into short, 20-25 nucleotide (nt) small RNA duplexes, of which one strand is bound to Argonaute (AGO) proteins to form an RNA- induced silencing complex (RISC).
  • RISC RNA- induced silencing complex
  • This silencing complex uses the small RNA as a guide to find and bind to complementary single- stranded RNA, where the AGO protein cleaves the RNA resulting in its degradation.
  • miRNAs are 20-24 nt small RNAs processed in the nucleus by Dicer-like 1 (DCL1) from short stem- loop precursor RNAs that are transcribed by RNA polymerase II from MIR genes.
  • DCL1 Dicer-like 1
  • tasiRNAs are phased siRNAs of primarily 21 nt in size derived from DCL4 processing of long dsRNA synthesized by RNA-dependent RNA polymerase 6 (RDR6) from miRNA-cleaved TAS RNA fragment.
  • RDR6 RNA-dependent RNA polymerase 6
  • the 24-nt rasiRNAs are processed by DCL3, and the precursor dsRNA is generated by the combined function of plant-specific DNA-dependent RNA polymerase IV (PolIV) and RDR2 from repetitive DNA in the genome.
  • the exosiRNA pathway overlaps with the tasiRNA and rasiRNA pathways and both DCL4 and DCL3 are involved in exosiRNA processing.
  • DCL1, DCL3 and DCL4 the model plant Arabidopsis thaliana and other higher plants encodes DCL2 or equivalent, which generates 22-nt siRNAs including 22-nt exosiRNAs, and plays a key role in systemic and transitive gene silencing in plants.
  • RNA-directed DNA methylation RdDM
  • RNA silencing induced by dsRNA has been extensively exploited to reduce gene activity in various eukaryotic systems, and a number of gene silencing technologies has been developed. Different organisms are often amenable to different gene silencing approaches. For instance, long dsRNA (at least 100 basepairs in length) is less suited to inducing RNA silencing in mammalian cells due to dsRNA-induced interferon responses, and so shorter dsRNAs (less than 30 basepairs) are generally used in mammalian cells, whereas in plants hairpin RNA (hpRNA) with a long dsRNA stem is highly effective.
  • hpRNA hairpin RNA
  • a hpRNA transgene construct typically consists of an inverted repeat made up of fully complementary sense and antisense sequences of a target gene sequence (which when transcribed form the dsRNA stem of hpRNA) separated by a spacer sequence (forming the loop of hpRNA), which is inserted between a promoter and a transcription terminator for expression in plant cells.
  • the spacer sequence functions to stabilize the inverted-repeat DNA in bacteria during construct preparation.
  • hpRNA transgenes have been widely used to knock down gene expression, modify metabolic pathways and enhance disease and pest resistance in plants for crop improvement, and many successful applications of the technology in crop improvement have now been reported (Guo et ah, 2016; Kim et ah, 2019).
  • hpRNA transgenes are subject to self-induced transcriptional repression compromising the stability and efficacy of target gene silencing. While all transgenes are potentially subject to position or copy number-dependent transcriptional silencing, hpRNA transgenes are unique as they generate siRNAs that can direct DNA methylation to their own sequence via the RdDM pathway, and this has the potential to cause transcriptional self-silencing.
  • RNA molecules which include one or more double-stranded RNA regions which comprise multiple non-canonically basepaired nucleotides or non-basepaired nucleotides, or both, including forms which have two or more loop sequences, herein called loop-ended dsRNA (ledRNA).
  • RNA molecules have one or more of the following features; they are easily synthesized, they accumulate to higher levels in plant cells upon transcription of the genetic constructs encoding them, they more readily form a dsRNA structure and induce efficient silencing of target RNA molecules in plant cells, and they may form circular RNA molecules upon processing in plant cells.
  • RNA molecules applied either endogenously, or preferably exogenously to plant cells at an earlier time, for example to seeds that give rise to the plants.
  • the RNA molecules may reduce or abolish the function of one or more genes involved in the timing of flowering, for example a repressor of flowering, and so promote flowering.
  • the present disclosure also provides a method of influencing the timing of flowering of a plant. This may be used to reduce or suppress activity of a gene with ability to influence a flowering characteristic through reduced expression of the gene by targeting its RNA transcripts. This modulation may be used to promote synchronous flowering of male and female parent lines in hybrid seed production, for example. Another use is to advance or retard flowering according to the variation of weather, or to extend or reduce the growing season.
  • the activity of the plant gene is preferably reduced as a result of under expression within at least some cells of the plant.
  • RNA molecules of the invention are advantageous in this context.
  • the present invention provides an RNA molecule comprising a first RNA component, a second RNA component which is covalently linked to the first RNA component and, optionally, one or more or all of (i) a linking ribonucleotide sequence which covalently links the first and second RNA components, (ii) a 5’ leader sequence and (iii) a 3’ trailer sequence, wherein the first RNA component consists of, in 5’ to 3’ order, a first 5’ ribonucleotide, a first RNA sequence and a first 3’ ribonucleotide, wherein the first 5’ and 3’ ribonucleotides basepair with each other in the first RNA component, wherein the first RNA sequence comprises a first sense ribonucleotide sequence of at least 20 contiguous ribonucleotides, a first loop sequence of at least 4 ribonucleotides and a first antisense ribonucleo
  • the present invention provides an RNA molecule comprising a first RNA component, a second RNA component which is covalently linked to the first RNA component and, optionally, one or more or all of (i) a linking ribonucleotide sequence which covalently links the first and second RNA components, (ii) a 5’ leader sequence and (iii) a 3’ trailer sequence, wherein the first RNA component consists of, in 5’ to 3’ order, a first 5’ ribonucleotide, a first RNA sequence and a first 3’ ribonucleotide, wherein the first 5’ and 3’ ribonucleotides basepair, wherein the first RNA sequence comprises a first sense ribonucleotide sequence, a first loop sequence of at least 4 ribonucleotides and a first antisense ribonucleotide sequence, wherein the first sense ribonucleotide sequence and first antisense ribonucleo
  • At least 20 contiguous ribonucleotides of the first antisense ribonucleotide sequence are all capable of basepairing to nucleotides of the first region of the target RNA molecule.
  • the first sense ribonucleotide sequence is linked covalently to the first 5’ ribonucleotide without any intervening nucleotides, or the first antisense ribonucleotide sequence is linked covalently to the first 3’ ribonucleotide without any intervening nucleotides, or both.
  • the RNA molecule comprises the linking ribonucleotide sequence, wherein the linking ribonucleotide sequence is less than 20 ribonucleotides. In an embodiment, the linking ribonucleotide sequence hybridizes to the target RNA molecule. In an embodiment, the linking ribonucleotide sequence is identical to a portion of the complement of the target RNA molecule. In another embodiment, the linking ribonucleotide sequence is between 1 and 10 ribonucleotides in length.
  • the RNA molecule comprises two or more sense ribonucleotide sequences, and antisense ribonucleotide sequences fully based paired thereto, which are identical in sequence to a region of a target RNA molecule.
  • the two or more sense ribonucleotide sequences are identical in sequence to different regions of the same target RNA molecule.
  • the two or more sense ribonucleotide sequences are identical in sequence to a region of different target RNA molecules.
  • the two or more sense ribonucleotide sequences have no intervening loop sequences.
  • the RNA molecule comprises two or more antisense ribonucleotide sequences, and sense ribonucleotide sequences fully based paired thereto, which are each complementary to a region of a target RNA molecule.
  • the two or more antisense ribonucleotide sequences are complementary to different regions of the same target RNA molecule.
  • the second of the two or more antisense ribonucleotide sequences are complementary to region of a different target RNA molecule than the first of the two or more antisense ribonucleotide sequences.
  • the two or more sense ribonucleotide sequences have no intervening loop sequences.
  • the RNA molecule is a single strand of ribonucleotides having a 5’ end, at least one sense ribonucleotide sequence which is at least 21 nucleotides in length, an antisense ribonucleotide sequence which is fully base paired with each sense ribonucleotide sequence over at least 21 contiguous nucleotides, at least two loop sequences and a 3’ end.
  • the RNA molecule is a single strand of ribonucleotides having a 5’ end, at least one sense ribonucleotide sequence which is at least 21 nucleotides in length, an antisense ribonucleotide sequence which is fully base paired with each sense ribonucleotide sequence over at least 21 contiguous nucleotides, at least two loop sequences and a 3’ end.
  • the RNA molecule is a single strand of ribonucleotides comprising a 5’ end, the first RNA component comprising a first sense ribonucleotide sequence which is at least 21 nucleotides in length, at least one loop sequence, a first antisense ribonucleotide sequence which hybridises with the first sense ribonucleotide sequence over a length of at least 21 contiguous nucleotides, and the second RNA component comprising a second sense ribonucleotide sequence which is at least 21 nucleotides in length, a loop sequence, a second antisense ribonucleotide sequence which hybridises with the second sense ribonucleotide sequence over a length of at least 21 contiguous nucleotides, and a 3’ end, wherein the RNA molecule has only one 5’ end and only one 3’ end.
  • the ribonucleotide at the 5’ end and the ribonucleotide at the 3’ end are adjacent, each base paired and are not directly covalently bonded.
  • the RNA molecule comprises a first antisense ribonucleotide sequence which hybridizes to a first region of a target RNA, a second antisense ribonucleotide sequence which hybridizes to a second region of a target RNA, the second region of the target RNA being different to the first region of the target RNA, and the RNA molecule comprising only one sense ribonucleotide sequence which hybridizes to the target RNA, wherein the two antisense sequences are not contiguous in the RNA molecule.
  • the RNA molecule comprises a first sense ribonucleotide sequence which is at least 60% identical to a first region of a target RNA, a second sense ribonucleotide sequence which is at least 60% identical to a second region of a target RNA, the second region of the target RNA being different to the first region of the target RNA, and the RNA molecule comprising only one antisense ribonucleotide sequence which hybridizes to the target RNA, wherein the two sense sequences are not contiguous in the RNA molecule.
  • the RNA molecule has the 5’ leader sequence.
  • the RNA molecule has the 3’ trailer sequence.
  • each ribonucleotide is covalently linked to two other nucleotides.
  • at least one or all of the loop sequences are longer than 20 nucleotides.
  • the RNA molecules has none, or one, or two or more bulges, or a double-stranded region of the RNA molecule comprises one, or two, or more nucleotides which are not basepaired in the double- stranded region.
  • the RNA molecule has three, four or more loops.
  • the RNA molecule only has two loops.
  • all of the loops are between 4 and 1,000 ribonucleotides, or between 4 and 200 ribonucleotides, in length.
  • all of the loops are between 4 and 50 ribonucleotides in length.
  • each loop is between 20 and 30 ribonucleotides in length.
  • the at least 20 contiguous ribonucleotides of the first antisense ribonucleotide sequence are all capable of basepairing to nucleotides of the first region of the target RNA molecule.
  • basepairing may be canonical or non-canonical, for example with at least some G:U basepairs. Independently for each G:U basepair, the G may be in the first region of the target RNA molecule or preferably in the first antisense ribonucleotide sequence.
  • the at least 20 contiguous ribonucleotides of the first antisense ribonucleotide sequence that are all capable of basepairing to nucleotides of the first region of the target RNA molecule do so by a canonical base pair.
  • not all of the at least 20 contiguous ribonucleotides of the first antisense ribonucleotide sequence basepair to nucleotides of the first region of the target RNA molecule.
  • 1, 2, 3, 4 or 5 of the at least 20 contiguous ribonucleotides of the first antisense ribonucleotide sequence are not basepaired to the first region of the target RNA molecule.
  • the first sense ribonucleotide sequence is linked covalently to the first 5’ ribonucleotide without any intervening nucleotides, or the first antisense ribonucleotide sequence is linked covalently to the first 3’ ribonucleotide without any intervening nucleotides, or both.
  • the RNA molecule comprises one or more linking ribonucleotide sequence, wherein the linking ribonucleotide sequence is related in sequence to the target RNA molecule, either identical at least in part to a region of the target RNA molecule or to its complement.
  • the linking ribonucleotide sequence together with sense sequences in the first and second RNA components form part of one contiguous sense sequence, or together with antisense sequences in the first and second RNA components form part of one contiguous antisense sequence.
  • the RNA molecule comprises the linking ribonucleotide sequence, wherein the linking ribonucleotide sequence is less than 20 ribonucleotides.
  • the linking ribonucleotide sequence hybridizes to the target RNA molecule. In an embodiment, the linking ribonucleotide sequence is identical to a portion of the complement of the target RNA molecule. In an embodiment, the linking ribonucleotide sequence is between 1 and 50, or between 1 and 10 ribonucleotides, in length.
  • the RNA molecule comprises two or more sense ribonucleotide sequences, and antisense ribonucleotide sequences fully based paired thereto, which are identical in sequence to a region of a target RNA molecule.
  • the two or more sense ribonucleotide sequences are identical in sequence to different regions of the same target RNA molecule.
  • the two or more sense ribonucleotide sequences are identical in sequence to a region of different target RNA molecules.
  • the two or more sense ribonucleotide sequences have no intervening loop sequences, i.e. they are contiguous relative to the target RNA molecule.
  • the RNA comprises two or more antisense ribonucleotide sequences, and sense ribonucleotide sequences fully based paired thereto, which are each complementary to a region of a target RNA molecule.
  • the two or more antisense ribonucleotide sequences are complementary to different regions of the same target RNA molecule.
  • the second of the two or more antisense ribonucleotide sequences are complementary to region of a different target RNA molecule than the first of the two or more antisense ribonucleotide sequences.
  • the RNA molecule is a single strand of ribonucleotides having a 5’ end, at least one sense ribonucleotide sequence which is at least 21 nucleotides in length, an antisense ribonucleotide sequence which is fully base paired with each sense ribonucleotide sequence over at least 21 contiguous nucleotides, at least two loop sequences and a 3’ end.
  • the RNA molecule is a single strand of ribonucleotides having a 5’ end, at least one sense ribonucleotide sequence which is at least 21 nucleotides in length, an antisense ribonucleotide sequence which is fully base paired with each sense ribonucleotide sequence over at least 21 contiguous nucleotides, at least two loop sequences and a 3’ end.
  • the RNA molecule is a a single strand of ribonucleotides comprising a 5’ end, the first RNA component comprising a first sense ribonucleotide sequence which is at least 21 nucleotides in length, at least one loop sequence, a first antisense ribonucleotide sequence which hybridises with the first sense ribonucleotide sequence over a length of at least 21 contiguous nucleotides, and the second RNA component comprising a second sense ribonucleotide sequence which is at least 21 nucleotides in length, a loop sequence, a second antisense ribonucleotide sequence which hybridises with the second sense ribonucleotide sequence over a length of at least 21 contiguous nucleotides, and a 3’ end, wherein the RNA molecule has only one 5’ end and only one 3’ end.
  • the ribonucleotide at the 5’ end and the ribonucleotide at the 3’ end are adjacent, each base paired and are not directly covalently bonded.
  • the RNA molecule comprises a first antisense ribonucleotide sequence which hybridizes to a first region of a target RNA, a second antisense ribonucleotide sequence which hybridizes to a second region of a target RNA, the second region of the target RNA being different to the first region of the target RNA, and the RNA molecule comprising only one sense ribonucleotide sequence which hybridizes to the target RNA, wherein the two antisense sequences are not contiguous in the RNA molecule.
  • the RNA molecule comprises a first sense ribonucleotide sequence which is at least 60% identical to a first region of a target RNA, a second sense ribonucleotide sequence which is at least 60% identical to a second region of a target RNA, the second region of the target RNA being different to the first region of the target RNA, and the RNA molecule comprising only one antisense ribonucleotide sequence which hybridizes to the target RNA, wherein the two sense sequences are not contiguous in the RNA molecule.
  • the RNA molecule has the 5’ leader sequence.
  • the RNA molecule has the 3’ trailer sequence.
  • each ribonucleotide is covalently linked to two other nucleotides.
  • the RNA molecule may be represented as a dumbbell shape ( Figure 1) but have a gap or nick in one part of the double-stranded structure.
  • At least one or all of the loop sequences are longer than 20 nucleotides.
  • the RNA molecules has none, or one, or two or more bulges, or a double-stranded region of the RNA molecule comprises one, or two, or more nucleotides which are not basepaired in the double- stranded region.
  • the RNA molecules has three, four or more loops.
  • the RNA molecules has only has two loops.
  • the target RNA is in a plant cell.
  • plants cells include, but are not limited to, those from Arabidopsis, corn, canola, cotton, soybean, alfalfa, lettuce, wheat, barley, rice, legume, Medicago truncatula, sugarbeet or rye.
  • the plant cell may be from a legume such as alfalfa or clover, a leafy vegetable e.g. lettuce, or a grass e.g. turfgrass.
  • the RNA molecule is present in a plant cell.
  • the RNA molecule of the invention is produced/expressed in a cell, such as for example a bacterial cell or other microbial cell, which is different to the cell comprising the target RNA.
  • the microbial cell is a cell in which the RNA molecule is produced by transcription from a genetic construct encoding the RNA molecule, wherein the RNA molecule is substantially, or preferably predominantly, not processed in the microbial cell by cleavage within one or more loop sequences, one or more dsRNA regions, or both.
  • the microbial cell is a yeast cell or another fungal cell which does not have a Dicer enzyme.
  • a greatly preferred cell for production of the RNA molecule is a Saccharomyces cerevisiae cell.
  • the microbial cell may be living, or may have been killed by some treatment such as heat treatment, or may be in the form of a dried powder.
  • at least one or all of the loop sequences of the RNA molecule are longer than 20 nucleotides.
  • at least one of the loops of the RNA molecule is between 4 and 1,200 ribonucleotides in length, or between 4 and 1000 ribonucleotides in length.
  • all of the loops are between 4 and 1,000 ribonucleotides in length.
  • At least one of the loops of the RNA molecule is between 4 and 200 ribonucleotides in length. In an even more preferred embodiment, all of the loops are between 4 and 200 ribonucleotides in length. In an even more preferred embodiment, at least one of the loops of the RNA molecule is between 4 and 50 ribonucleotides in length. In a most preferred embodiment, all of the loops are between 4 and 50 ribonucleotides in length. In embodiments, the minimum length of the loop is 20 nucleotides, 30 nucleotides, 40 nucleotides, or 50 nucleotides. In an embodiment, each loop of the RNA molecule is independently between 20 and 50 ribonucleotides, or between 20 and 40 ribonucleotides or between 20 and 30 ribonucleotides in length.
  • the target RNA encodes a protein.
  • the RNA molecule may comprise a region of a nucleotide sequence set forth in SEQ ID NO: 146, SEQ ID NO: 147, or SEQ ID NOs:151-152 (wheat), SEQ ID NOs:154-155 (barley), SEQ ID NOs:156-164 (rice), SEQ ID NOs: 165-178 (maize), SEQ ID NOs: 179-185 ( Brassica napus), SEQ ID NOs:186-187 and SEQ ID NO:210 ( Medicago truncatula), SEQ ID NOs:188-190 (alfalfa), SEQ ID NOs: 191-204 (soybean), SEQ ID N0s:205-207 (sugarbeet), SEQ ID N0s:208-209 ( Brassica rapa), SEQ ID NOs:211-220 (onion) and SEQ ID NOs:221- 228 (lettuce), or a complement (antisense) of a region of the
  • the RNA molecule of the invention comprises a sense and an antisense sequence from a region of an RNA transcript from a gene whose cDNA corresponds to one of the SEQ ID NOs listed above, or a nucleotide sequence 95% or preferably 99% identical thereto.
  • Such sequence is preferably derived from the RNA transcript of a naturally occurring homolog of the gene in that plant species.
  • RNA molecules of the invention may comprise a a region of a nucleotide sequence set forth in SEQ ID NO: 146, SEQ ID NO: 147 or SEQ ID NOs: 151-228.
  • the second RNA component is characterised in that: i) the second sense ribonucleotide sequence consists of at least 20 contiguous ribonucleotides covalently linked, in 5’ to 3’ order, the second 5’ ribonucleotide, a third RNA sequence and a third 3’ ribonucleotide, ii) the second antisense ribonucleotide sequence consists of at least 20 contiguous ribonucleotides covalently linked, in 5’ to 3’ order, a third 5’ ribonucleotide, a fourth RNA sequence and the second 3’ ribonucleotide, iii) the second 5’ ribonucleotide basepairs with the second 3’ ribonucleotide, iv) the third 3’ ribonucleotide basepairs with the third 5’ ribonucleotide, wherein the chimeric
  • the asRNA molecules produced from the second antisense sequence are capable of reducing expression of the target RNA, either without or in combination with asRNAs produced from the first antisense sequence of the first RNA component. It is more preferred that between 5% and 40% of the ribonucleotides of the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence, and/or the second sense ribonucleotide sequence and the second antisense ribonucleotide sequence, and/or every sense ribonucleotide sequence and its corresponding antisense ribonucleotide sequence which hybridise, in total, are either basepaired in a non- canonical basepair or are not basepaired, and/or the dsRNA region formed between the complementary sense and antisense sequences does not comprise 20 contiguous canonical basepairs.
  • ribonucleotides of a sense ribonucleotide sequence and its corresponding antisense ribonucleotide sequence are either basepaired in a non-canonical basepair or are not basepaired.
  • ribonucleotides of the dsRNA region(s) in the RNA molecule are basepaired in non-canonical basepairs and all of the other ribonucleotides of the dsRNA region(s) in the RNA molecule are basepaired in canonical basepairs.
  • At least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% of the non-canonical basepairs in the first or second dsRNA region, or all dsRNA regions in total, are G:U basepairs.
  • the first and second antisense ribonucleotide sequences preferably every antisense ribonucleotide sequence in the RNA molecule, comprises a sequence of at least 20 contiguous ribonucleotides which is at least 50% identical in sequence to a region of the complement of the target RNA molecule, preferably at least 60% identical, more preferably at least 70% identical, even more preferably at least 80% identical, most preferably at least 90% identical or 100% identical to the region of the complement of the target RNA molecule, or both (a) and (b).
  • the present invention provides a chimeric ribonucleic acid (RNA) molecule, comprising a double-stranded RNA (dsRNA) region which comprises a first sense ribonucleotide sequence of at least 20 contiguous nucleotides in length and a first antisense ribonucleotide sequence of at least 20 contiguous nucleotides in length, whereby the first sense ribonucleotide sequence and the first antisense ribonucleotide sequences are capable of hybridising to each other to form the dsRNA region, wherein i) the first sense ribonucleotide sequence consists of, covalently linked in 5’ to 3’ order, a first 5’ ribonucleotide, a first RNA sequence and a first 3’ ribonucleotide, ii) the first antisense ribonucleotide sequence consists of, covalently linked in 5’ to 3’ order,
  • the first sense ribonucleotide sequence is covalently linked to the first antisense ribonucleotide sequence by a first linking ribonucleotide sequence which comprises a loop sequence of at least 4 nucleotides, or between 4 and 1,000 ribonucleotides, or between 4 and 200 ribonucleotides, or between 4 and 50 ribonucleotides, or at least 10 nucleotides, or between 10 and 1,000 ribonucleotides, or between 10 and 200 ribonucleotides, or between 10 and 50 ribonucleotides, in length, whereby the first linking ribonucleotide sequence is covalently linked to either the second 3’ ribonucleotide and the first 5’ ribonucleotide or, preferably, to the first 3’ ribonucleotide and the second 5’ ribonucleotide, so that the sequences are comprised in a single, con
  • the first linking ribonucleotide sequence is covalently linked to either the second 3’ ribonucleotide and the first 5’ ribonucleotide or, preferably, to the first 3’ ribonucleotide and the second 5’ ribonucleotide, so that the sequences are comprised in a single, contiguous strand of RNA.
  • the loop sequence in the chimeric RNA molecule comprises one or more binding sequences which are complementary to an RNA molecule which is endogenous to the plant cell, and/or the loop sequence in the RNA molecule comprises an open reading frame which encodes a polypeptide or a functional polynucleotide.
  • a hairpin RNA hpRNA
  • hpRNA hairpin RNA
  • the ribonucleotides of the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence of the dsRNA in total, are basepaired in non-canonical basepairs, preferably G:U basepairs.
  • the first antisense ribonucleotide sequence is fully complementary to a region of the target RNA.
  • the first sense ribonucleotide sequence is different in sequence to the region of the target RNA by the substitution of C nucleotides in the region of the target RNA with U nucleotides in the hpRNA.
  • Such molecules are exemplified in the hairpin RNAs comprising G:U basepairs in Examples 6-11.
  • the length of the first antisense ribonucleotide sequence is 20 to about 1000 nucleotides, or 20 to about 500 nucleotides, or other lengths as described herein.
  • the hpRNA is produced in, or introduced into, a plant cell.
  • the target RNA may be a transcript of an endogenous gene in the plant cell.
  • the first antisense ribonucleotide sequence is fully complementary to a region of the target RNA and the first sense ribonucleotide sequence is different in sequence to the region of the target RNA by the substitution of C nucleotides in the region of the target RNA with U nucleotides.
  • the chimeric RNA molecule comprises a second sense ribonucleotide sequence and the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence are linked by a first linking ribonucleotide sequence comprising a loop sequence of at least 4 nucleotides in length, whereby the first linking ribonucleotide sequence is covalently linked to the first 3’ ribonucleotide and the second 5’ ribonucleotide, and the RNA molecule further comprises a second linking ribonucleotide sequence which comprises a loop sequence of at least 4 nucleotides in length and which is covalently linked to the second 3’ ribonucleotide and the second sense ribonucleotide sequence, thereby forming an ledRNA structure.
  • the chimeric RNA molecule comprises a second antisense ribonucleotide sequence and the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence are linked by a first linking ribonucleotide sequence comprising a loop sequence of at least 4 nucleotides in length, whereby the first linking ribonucleotide sequence is covalently linked to the second 3’ ribonucleotide and the first 5’ ribonucleotide, and the RNA molecule further comprises a second linking ribonucleotide sequence which comprises a loop sequence of at least 4 nucleotides in length and which is covalently linked to the second 3’ ribonucleotide and the second antisense ribonucleotide sequence.
  • the chimeric RNA molecule comprises a second sense ribonucleotide sequence and a second antisense ribonucleotide sequence, wherein the second sense ribonucleotide sequence and the second antisense ribonucleotide sequences are capable of hybridising to each other to form a second dsRNA region, and the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence are linked by a first linking ribonucleotide sequence comprising a loop sequence of at least 4 nucleotides in length, whereby the first linking ribonucleotide sequence is covalently linked to the first 3’ ribonucleotide and the second 5’ ribonucleotide, and the RNA molecule further, or optionally, comprises a second linking ribonucleotide sequence which comprises a loop sequence of at least 4 nucleotides in length and which is covalently linked to the second 3
  • the chimeric RNA molecule comprises a second sense ribonucleotide sequence and a second antisense ribonucleotide sequence and the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence are linked by a first linking ribonucleotide sequence comprising a loop sequence of at least 4 nucleotides in length, whereby the first linking ribonucleotide sequence is covalently linked to the second 3’ ribonucleotide and the first 5’ ribonucleotide, and the RNA molecule further comprises a second linking ribonucleotide sequence which comprises a loop sequence of at least 4 nucleotides in length and which is covalently linked to the first 3’ ribonucleotide and the second antisense ribonucleotide sequence, or which covalently links the second sense ribonucleotide sequence and the second antisense ribonucleotide sequence
  • the chimeric RNA molecule comprises a second sense ribonucleotide sequence and a second antisense ribonucleotide sequence and the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence are linked by a first linking ribonucleotide sequence comprising a loop sequence of at least 4 nucleotides in length, whereby the first linking ribonucleotide sequence is covalently linked to the second 3’ ribonucleotide and the first 5’ ribonucleotide, and the RNA molecule further comprises a second linking ribonucleotide sequence which comprises a loop sequence of at least 4 nucleotides in length and which is covalently linked to the first 3’ ribonucleotide and the second antisense ribonucleotide sequence, or which covalently links the second sense ribonucleotide sequence and the second antisense ribonucleotide sequence
  • the second sense ribonucleotide sequence and the second antisense ribonucleotide sequence each comprise at least 20 contiguous nucleotides in length.
  • the first and second sense ribonucleotide sequences are covalently linked by an intervening ribonucleotide sequence which is unrelated in sequence to the target RNA molecule, or which is related in sequence to the target RNA molecule, or the first and second sense ribonucleotide sequences are covalently linked without an intervening ribonucleotide sequence.
  • the first and second antisense ribonucleotide sequences are covalently linked by an intervening ribonucleotide sequence which is unrelated in sequence to the complement of a target RNA molecule, or which is related in sequence to the complement of a target RNA molecule, or the first and second antisense ribonucleotide sequences are covalently linked without an intervening ribonucleotide sequence.
  • first and second sense ribonucleotide sequences may form one contiguous sense ribonucleotide region having at least 50% identity in sequence to a target RNA molecule.
  • first and second antisense sense ribonucleotide sequences may form one contiguous antisense ribonucleotide region having at least 50% identity in sequence to the complement of a target RNA molecule.
  • the RNA molecule comprises a first sense ribonucleotide sequence which is at least 60% identical to a first region of a target RNA, a second sense ribonucleotide sequence which is at least 60% identical to a second region of a target RNA, the second region of the target RNA being different to the first region of the target RNA, and the RNA molecule comprising only one antisense ribonucleotide sequence which hybridizes to the target RNA, wherein the two sense sequences are not contiguous in the RNA molecule.
  • the first and second regions of the target RNA are contiguous in the target RNA molecule. Alternatively, they are not contiguous.
  • the first and second sense ribonucleotide sequences are each, independently, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to the respective region of target RNA i.e. the first sense sequence may be at least 70% identical to its target region and the second sequence at least 80% identical to its target sequence, etc.
  • ribonucleotides of the second sense ribonucleotide sequence and the second antisense ribonucleotide sequence in total, are either basepaired in a non-canonical basepair or are not basepaired, preferably basepaired in G:U basepairs, wherein the second dsRNA region does not comprise 20 contiguous canonical basepairs, and wherein the RNA molecule is capable of being processed in a eukaryotic cell or in vitro whereby the second antisense ribonucleotide sequence is cleaved to produce short antisense RNA (asRNA) molecules of 20-24 ribonucleotides in length.
  • asRNA short antisense RNA
  • each linking ribonucleotide sequence is independently between 4 and about 2000 nucleotides in length, preferably between 4 and about 1200 nucleotides in length, more preferably between 4 and about 200 nucleotides in length and most preferably between 4 and about 50 nucleotides in length.
  • the chimeric RNA molecule further comprises a 5’ leader sequence or a 3’ trailer sequence, or both.
  • the present invention provides a chimeric RNA molecule comprising a first RNA component and a second RNA component which is covalently linked to the first RNA component, wherein the first RNA component comprises a first double-stranded RNA (dsRNA) region, which comprises a first sense ribonucleotide sequence and a first antisense ribonucleotide sequence which are capable of hybridising to each other to form the first dsRNA region, and a first intervening ribonucleotide sequence of at least 4 nucleotides which covalently links the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence, wherein the second RNA component comprises a second sense ribonucleotide sequence, a second antisense ribonucleotide sequence and a second intervening rib
  • dsRNA double
  • the chimeric RNA molecule or at least some of the asRNA molecules, or both are capable of reducing the expression or activity of a target RNA molecule which modulates plant flowering, or
  • the first antisense ribonucleotide sequence comprises a sequence of at least 20 contiguous ribonucleotides which is at least 50% identical in sequence, preferably at least 90% or 100% identical in sequence, to a region of the complement of the target RNA molecule, or (c) both (a) and (b).
  • the at least 20 contiguous ribonucleotides of the first antisense ribonucleotide sequence are all capable of basepairing to nucleotides of a first region of the target RNA molecule.
  • the chimeric RNA molecule comprises two or more antisense ribonucleotide sequences, and sense ribonucleotide sequences based paired thereto, which antisense sequences are each complementary, preferably fully complementary, to a region of a target RNA molecule.
  • the regions of the target RNA molecule to which they are complementary may or may not be contiguous in the target RNA molecule.
  • the two or more antisense ribonucleotide sequences are complementary to different regions of the same target RNA molecule.
  • the two or more antisense ribonucleotide sequences are complementary to regions of different target RNA molecules.
  • the two or more antisense ribonucleotide sequences have no intervening loop sequences, i.e. they are contiguous relative to the complement of the target RNA molecule.
  • one or both of the two or more antisense ribonucleotide sequences and sense ribonucleotide sequences basepair along their full length through canonical basepairs, or through some canonical and some non- canonical basepairs, preferably G:U basepairs.
  • the RNA molecule may comprise a 5 ’-leader sequence and/or a 3 ’-trailer sequence.
  • the chimeric RNA molecule comprises a hairpin RNA (hpRNA) structure having a 5’ end, a sense ribonucleotide sequence which is at least 21 nucleotides in length, an antisense ribonucleotide sequence which is fully base paired with the sense ribonucleotide sequence over at least 21 contiguous nucleotides, an intervening loop sequence and a 3’ end.
  • hpRNA hairpin RNA
  • the RNA molecule may comprise a 5 ’-leader sequence and/or a 3 ’-trailer sequence.
  • the chimeric RNA molecule comprises a single strand of ribonucleotides having a 5’ end, at least one sense ribonucleotide sequence which is at least 21 nucleotides in length, an antisense ribonucleotide sequence which is fully base paired with each sense ribonucleotide sequence over at least 21 contiguous nucleotides, at least two loop sequences and a 3’ end.
  • the order 5’ to 3’ may be the sense ribonucleotide sequence and then the antisense ribonucleotide sequence, or vice versa.
  • the ribonucleotide at the 5’ end and the ribonucleotide at the 3’ end are adjacent, each base paired and are not directly covalently bonded, see for example Figure 1.
  • between about 15% and about 30%, or between about 16% and about 25%, of the ribonucleotides of the sense ribonucleotide sequence and the antisense ribonucleotide sequence, in total, are either basepaired in a non-canonical basepair or are not basepaired, preferably basepaired in non-canonical basepairs, more preferably basepaired in G:U basepairs.
  • At least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% of the non- canonical basepairs are G:U basepairs.
  • less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1% or none, of the ribonucleotides in the dsRNA region are not basepaired.
  • every one in four to every one in six ribonucleotides in the dsRNA region form a non-canonical basepair or are not basepaired, preferably form a G:U basepair.
  • the dsRNA region does not comprise 8 contiguous canonical basepairs.
  • the dsRNA region comprises at least 8 contiguous canonical basepairs, preferably at least 8 but not more than 12 contiguous canonical basepairs.
  • all of the ribonucleotides in the dsRNA region, or in each dsRNA region, are base-paired with a canonical basepair or a non-canonical basepair.
  • one or more ribonucleotides of the sense ribonucleotide sequence or one or more ribonucleotides of the antisense ribonucleotide sequence, or both are not basepaired.
  • the antisense RNA sequence is less than 100% identical, or between about 80% and 99.9% identical, or between about 90% and 98% identical, or between about 95% and 98% identical, in sequence to the complement of a region of the target RNA molecule.
  • the antisense RNA sequence is 100% identical in sequence to a region of the target RNA molecule.
  • the sense and/or antisense ribonucleotide sequence preferably both, is at least 50, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1,000, or about 100 to about 1,000, or 20 to about 1000 nucleotides, or 20 to about 500 nucleotides, in length.
  • the number of ribonucleotides in the sense ribonucleotide sequence is between about 90% and about 110% of the number of ribonucleotides in the antisense ribonucleotide sequence.
  • the number of ribonucleotides in the sense ribonucleotide sequence is the same as the number of ribonucleotides in the antisense ribonucleotide sequence.
  • the chimeric RNA molecule further comprises a 5’ extension sequence which is covalently linked to the first 5’ ribonucleotide or a 3’ extension sequence which is covalently linked to the second 3’ ribonucleotide, or both.
  • the chimeric RNA molecule further comprises a 5’ extension sequence which is covalently linked to the second 5’ ribonucleotide or a 3’ extension sequence which is covalently linked to the first 3’ ribonucleotide, or both.
  • the chimeric RNA molecule comprises two or more dsRNA regions which are the same or different.
  • RNA molecules when expressed in a plant cell more asRNA molecules are formed that are 22 and/or 20 ribonucleotides in length when compared to processing of an analogous RNA molecule which has a corresponding dsRNA region which is fully basepaired with canonical basepairs.
  • an RNA molecule of the first or second aspect is also a chimeric RNA meolceule of the third or fourth aspects.
  • the target RNA encodes VERNALIZATION 1 (VRN1), VERNALIZATION2 (VRN2),
  • the target RNA comprises a region of a nucleotide sequence set forth in any one or more of SEQ ID NO’s 146, 147, or 151 to 228 (where the T’s are replaced with U’s), or a complement (antisense) of the region of the sequence, or both the region and the complement, or a nucleotide sequence 95%, preferably, 99%, identical thereto (where the T’s are replaced with U’s).
  • the region is at least 15, at least 16, a least 17, at least 18, at least 19, at least 20 or at least 21 nucleotides in length.
  • target RNA is a gene transcript of the following from wheat, with Accession Nos of the genes or proteins in parentheses: VRN1/VRN-A1 (KR422423.1; SEQ ID NO:151); VRN2 (ZCCT1, TaVRN2-B; SEQ ID NO:145) (AAS58481.1); TaFT (Accession No. AY705794.1; SEQ ID NO: 152) or homologous genes in other species, preferably cereal species.
  • the target RNA is a gene transcript of the one of the following from barley: HvVRNl (AY896051; SEQ ID NO: 153), HvVRN2 (AY687931, AY485978; SEQ ID NO: 154) or HvFT (DQ898519; SEQ ID NO: 155), or homologous genes in other species, preferably cereal species.
  • the target RNA is a gene transcript of one of the following from canola, BnFLCl (AY036888, Bna.FLC.A10, BnaA10g22080D; SEQ ID NO: 179); BnFLC2 (AY036889; SEQ ID NO: 180); BnFLC3 (AY036890; SEQ ID NO:181); BnFLC4 (AY036891; SEQ ID NO:182); BnFLC5 (AY036892; SEQ ID NO: 183); BnFRI (BnaA03gl3320D; SEQ ID NO: 184); BnFT (BnaA02gl2130D; SEQ ID NO: 185) or homologous genes in other species. .
  • BnFLCl AY036888, Bna.FLC.A10, BnaA10g22080D; SEQ ID NO: 179
  • BnFLC2 AY036889; SEQ ID NO: 180
  • the target RNA is a gene transcript of one of the following from Arabidopsis, FRI (AT4G00650); FLC (AT5G10140); VRN1 (AT3G18990); VRN2 (AT4G16845); VIN3 (AT5G57380); FT (AT1G65480); SOC1 (AT2G45660); CO (constans) (AT5G15840); LFY (AT5G61850); API (AT1G69120) or homologous genes in other species.
  • FRI Arabidopsis
  • FLC AT5G10140
  • VRN1 AT3G18990
  • VRN2 AT4G16845
  • VIN3 AT5G57380
  • FT AT1G65480
  • SOC1 AT2G45660
  • CO constans
  • AT5G15840 LFY (AT5G61850); API (AT1G69120) or homologous genes in other species.
  • the target RNA is a gene transcript of one of the following from rice, OsPhyB (OSNPB_030309200; SEQ ID NO: 156); OsCol4 (HC084637; SEQ ID NO: 157); RFT1 (OSNPB_070486100; SEQ ID NO:158); OsSNB (OSNPB_070235800; SEQ ID NO:159); OsIDSl (Os03g0818800; SEQ ID NO: 160); OsGI (OSNPB_010182600; SEQ ID NO: 161), OsMADS50 (SEQ ID NO: 162), OsMADS55 (SEQ ID NO: 163) or OsLFY (SEQ ID NO: 164), or homologous genes in other species.
  • OsPhyB (OSNPB_030309200; SEQ ID NO: 156); OsCol4 (HC084637; SEQ ID NO: 157); RFT1 (OSNPB_070486100; SEQ ID NO:158); Os
  • the target RNA is a gene transcript of the one of the following from maize (Zea mays): ZmMADSl/ZmM5 (LOC542042, HM993639; SEQ ID NO:), PHYA1 (AY234826; SEQ ID NO: 166), PHYA2 (AY260865; SEQ ID NO: 167), PHYB1 (AY234827; SEQ ID NO: 168), PHYB2 (AY234828; SEQ ID NO: 169), PHYC1 (AY234829; SEQ ID NO: 170), PHYC2 (AY234830; SEQ ID NO: 171), ZmLD (AF166527; SEQ ID NO: 172), ZmFLl (AY179882; SEQ ID NO: 173), ZmFL2 (AY179881; SEQ ID NO: 174), DWARF8 (AF413203; SEQ ID NO: 175), ZmANl (L37750; SEQ ID NO:
  • the target RNA is a gene transcript of one of the following from Medicago truncatula, MtFTal (HQ721813; SEQ ID NO: 186); MtFTbl (HQ721815; SEQ ID NO: 187), MtYFL (BT053010, SEQ ID NO:210), MtSOCla (Medtr07g075870), MtSOClb (Medtr08g033250), MtSOClc (Medtr08g033220), or homologous genes in other species.
  • MtFTal HQ721813; SEQ ID NO: 186
  • MtFTbl HQ721815; SEQ ID NO: 187
  • MtYFL B053010, SEQ ID NO:210
  • MtSOCla Medtr07g075870
  • MtSOClb Medtr08g033250
  • MtSOClc Medtr08g033220
  • the target RNA is a gene transcript of one of the following from alfalfa ⁇ Medicago sativa ), MsFRI-L (SEQ ID NO: 188), MsSOCla (SEQ ID NO: 189), or MsFT (SEQ ID NO: 190), or homologous genes in other species.
  • the target RNA is a gene transcript of one of the following from soybean ⁇ Glycine max): encoded by the gene GLYMA_05G148700 with any one or more of the following transcript variants GmFLC-Xl (SEQ ID NO: 191), GmFLC-X2 (SEQ ID NO: 192) GmFLC-X3 (SEQ ID NO: 193), GmFLC-X4 (SEQ ID NO: 194), GmFLC-X5 (SEQ ID NO: 195), GmFLC-X6 (SEQ ID NO: 196), GmFLC-X7 (SEQ ID NO: 197), GmFLC-X8 (SEQ ID NO: 198), or GmFLC-X9 (SEQ ID NO: 199), or SUPPRESSOR OF FRI (SEQ ID NO:200), GmFRI (SEQ ID NO:201), GmFT2A (SEQ ID NO:202), GmPHYA3 (SEQ ID NO:203
  • the target RNA is a gene transcript of the following from sugarbeet (Beta vulgaris), BvBTCl (HQ709091, SEQ ID NO:205), preferably BvFTl (HM448909.1, SEQ ID NO:206) and/or BvFT2 (HM448911, SEQ ID NO:207), where RNAi-induced down-regulation of the BvFTl- BvFT2 module led to a strong delay in bolting after vernalization by several weeks, or BvFLl (DQ189214, DQ189215), or homologous genes in other species.
  • the target RNA is a gene transcript of one of the following genes from Brassica rapa, which may be turnip, cabbage, bok choi, turnip rape or related crucifers: BrFLC2 (AH012704, SEQ ID NO:208), BrFT (Bra004928) or BrFRI (HQ615935, SEQ ID NO:209), or homologous genes in other species.
  • the target RNA is a gene transcript of one of the following from cotton ⁇ Go sypium hirsutum: GhCO (Gorai.008G059900), GhFLC (Gorai.013G069000), GhFRI (Gorai.003Gl 18000), GhFT (Gorai.004G264600), GhLFY (Gorai.001G053900), GhPHYA (Gorai.007G292800, Gorai.013G203900), GhPHYB (Gorai.011G200200), GhSOCl (Gorai.008G 115200), GhVRNl (Gorai.002G006500, Gorai.005G240900,
  • the target RNA is a gene transcript of one of the following from onion (Allium cepa): AcGI (GQ232756, SEQ ID NO:211), AcFKF (GQ232754, SEQ ID NO:212), AcZTF (GQ232755, SEQ ID NO:213), AcCOL (GQ232751, SEQ ID NO:214), AcFTL (CF438000, SEQ ID NO:215), AcFTl (KC485348, SEQ ID NO:216), AcFT2 (KC485349, SEQ ID NO:217), AcFT6 (KC485353, SEQ ID NO:218), AcPHYA (GQ232753, SEQ ID NO:219), AcCOPl (CF451443, SEQ ID NO:220), or homologous genes in other species.
  • the target RNA is a gene transcript of one of the following from onion (Allium cepa): AcGI (GQ232756, SEQ ID NO:211), AcFKF (GQ232754, SEQ ID NO:212),
  • the target RNA is a gene transcript of one of the following from asparagus ( Asparagus officinalis ): FPA (LOC 109824259, LOC 109840062), TWIN SISTER of FT-like (LOC 109835987), MOTHER of FT (LOC 109844838), FCA-like (LOC109841154, LOC109821266), PHOTOPERIOD-INDEPENDENT EARLY
  • FLOWERING 1 (LOC109834006), FLOWERING LOCUS T-like (LOC 109830558, LOC109825338, LOC 109824462), Flowering locus K (LOC 109847537), Flowering time control protein FY (LOC109844014), flowering time control protein FCA-like (LOC 109842562), or homologous genes in other species.
  • the target RNA is a gene transcript of one of the following from lettuce ( Lactuca sativa ): LsFT (LOCI 11907824, SEQ ID NO:221), TFLl-like (LOCI 11903066, SEQ ID NO:222), TFL1 homolog 1-like (LOCI 11903054, SEQ ID NO:223), LsFLC (LOCI 11876490, JI588382, SEQ ID NO:224), LsSOCl-like (LOCI 11912847, SEQ ID NO:225, LOCI 11880753, SEQ ID NO:226, LOCI 11878575, SEQ ID NO:227), TsLFY (LC164345.1, XM_023888266.1, SEQ ID NO:228), or homologous genes in other species.
  • LsFT LOCI 11907824, SEQ ID NO:221
  • TFLl-like LOCI 11903066, SEQ ID NO:222
  • TFL1 homolog 1-like
  • the target RNA is a miRNA.
  • targets include, but are not limited to, miR-156 or miR-172.
  • the RNA molecule or chimeric RNA molecule reduces the time to flowering compared to an isogenic plant lacking the RNA molecule or chimeric RNA molecule.
  • the plant is Arabidopsis, com, canola, cotton, soybean, alfalfa, lettuce, wheat, barley, rice, legume, Medicago truncatula, sugarbeet or rye.
  • the plant is Arabidopsis, com, canola, cotton, soybean, wheat, barley, rice, legume, Medicago truncatula, sugarbeet or rye.
  • the plant may be from alfalfa or clover, a leafy vegetable e.g. lettuce, or a grass e.g. turfgrass.
  • the first and second regions of the target RNA are contiguous in the target RNA. Alternatively, they are not contiguous.
  • the RNA molecule or chimeric RNA molecule delays the time to flowering compared to an isogenic plant lacking the RNA molecule or chimeric RNA molecule.
  • the plant is a grass, where the target gene is a homolog of a cereal gene, as above.
  • the plant is genetically unmodified.
  • the RNA molecule comprises a 5’ leader sequence or 5’ extension sequence. In an embodiment, the RNA molecule comprises a 3’ trailer sequence or 3’ extension sequence. In a preferred embodiment, the RNA molecule comprises both the 5’ leader/extension sequence and the 3’ trailer/extension sequence.
  • At least one loop sequence in the RNA molecule comprises one or more binding sequences which are complementary to an RNA molecule which is endogenous to the plant cell, such as, for example, an miRNA or other regulatory RNA in the plant cell.
  • this feature may be in combination with any of the loop length features, non-canonical basepairing and any of the other features described above for the RNA molecule.
  • at least one loop sequence comprises multiple binding sequences for a miRNA, or binding sequences for multiple miRNAs, or both.
  • at least one loop sequence in the RNA molecule comprises an open reading frame which encodes a polypeptide or a functional polynucleotide.
  • the open reading frame is preferably operably linked to a translation initiation sequence, whereby the open reading frame is capable of being translated in a plant cell of interest.
  • the translation initiation sequence comprises, or is comprised in, an internal ribosome entry site (IRES).
  • IRES is preferably a plant IRES.
  • the translated polypeptide is preferably 50-400 amino acid residues in length, or 50-300 or 50-250, or 50-150 amino acid residues in length.
  • Such RNA molecules when produced in a plant cell, are capable of being processed to form circular RNA molecules comprising most or all of the loop sequence and which are capable of being translated to provide high levels of the polypeptide.
  • the RNA molecule has none, or one, or two or more bulges in a double-stranded region.
  • a bulge is a nucleotide, or two or more contiguous nucleotides, in the sense or antisense ribonucleotide sequence which is not basepaired in the dsRNA region and which does not have a mismatched nucleotide at the corresponding position in the complementary sequence in the dsRNA region.
  • the dsRNA region of the RNA molecule may comprise a sequence of more than 2 or 3 nucleotides within the sense or antisense sequence, or both, which loops out from the dsRNA region when the dsRNA structure forms. The sequence which loops out may itself form some internal basepairing, for example it may itself form a stem-loop structure.
  • the RNA molecule has none, or one, or two or more bulges in a double-stranded region.
  • a bulge is a nucleotide, or two or more contiguous nucleotides, in the sense or antisense ribonucleotide sequence which is not basepaired in the dsRNA region and which does not have a mismatched nucleotide at the corresponding position in the complementary sequence in the dsRNA region.
  • the dsRNA region of the RNA molecule may comprise a sequence of more than 2 or 3 nucleotides within the sense or antisense sequence, or both, which loops out from the dsRNA region when the dsRNA structure forms. The sequence which loops out may itself form some internal basepairing, for example it may itself form a stem-loop structure.
  • the RNA molecule has three, four or more loops. In a preferred embodiment, the RNA molecule has only two loops.
  • the first double- stranded region, or the first and second dsRNA region, or every dsRNA region, of the RNA molecule comprises one, or two, or more nucleotides which are not basepaired in the double-stranded region, or independently up to 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% of the nucleotides in the double- stranded region which are not basepaired.
  • about 12%, about 15%, about 18%, about 21%, about 24%, or between about 15% and about 30%, or preferably between about 16% and about 25%, of the ribonucleotides of the sense ribonucleotide sequence and its corresponding antisense ribonucleotide sequence, in total, that form a dsRNA region are either basepaired in a non-canonical basepair or are not basepaired.
  • At least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% of the non-canonical basepairs in a dsRNA region, or in all dsRNA regions in the RNA molecule, are G:U basepairs.
  • the G nucleotide in each G:U basepair may independently be in the sense ribonucleotide sequence or preferably in the antisense ribonucleotide sequence.
  • G nucleotides in the G:U basepairs of a dsRNA region preferably at least 50% are in the antisense ribonucleotide sequence, more preferably at least 60% or 70%, even more preferably at least 80% or 90%, and most preferably at least 95% of them are in the antisense ribonucleotide sequence in the dsRNA region.
  • This feature may apply independently to one or more or all of the dsRNA regions in the RNA molecule.
  • less than 25%, less than 20%, less than 15%, less than 10%, preferably less than 5%, more preferably less than 1% or most preferably none, of the ribonucleotides in the dsRNA region, or in all of the dsRNA regions in the RNA molecule in total are not basepaired.
  • every one in four to every one in six ribonucleotides in the dsRNA region, or in the dsRNA regions in total form a non-canonical basepair or are not basepaired within the RNA molecule.
  • the dsRNA region, or in the dsRNA regions in total do not comprise 10 or 9 or preferably 8 contiguous canonical basepairs.
  • the dsRNA region comprises at least 8 contiguous canonical basepairs, for example 8 to 12 or 8 to 14 or 8 to 10 contiguous canonical basepairs.
  • all of the ribonucleotides in the dsRNA region, or in all dsRNA regions in the RNA molecule are base-paired with a canonical basepair or a non-canonical basepair.
  • one or more ribonucleotides of the sense ribonucleotide sequence or one or more ribonucleotides of the antisense ribonucleotide sequence, or both, are not basepaired.
  • one or more ribonucleotides of each sense ribonucleotide sequence and one or more ribonucleotides of each antisense ribonucleotide sequence are not basepaired in the RNA molecule of the invention.
  • one or more or all of the antisense ribonucleotide sequences of the RNA molecule is less than 100% identical, or between about 80% and 99.9% identical, or between about 90% and 98% identical, or between about 95% and 98% identical, preferably between 98% and 99.9% identical, in sequence to the complement of a region of the target RNA molecule or to two such regions, which may or may not be contiguous in the target RNA molecule.
  • one or more of the antisense RNA sequences is 100% identical in sequence to a region of the complement of the target RNA molecule, for example to a region comprising 21, 23, 25, 27, 30, or 32 contiguous nucleotides.
  • the sense or antisense ribonucleotide sequence is at least 40, at least 50, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1,000, or about 100 to about 1,000, contiguous nucleotides in length.
  • the lengths of at least 100 nucleotides are preferred when using the RNA molecule in plant cells.
  • the number of ribonucleotides in the sense ribonucleotide sequence is between about 90% and about 110%, preferably between 95% and 105%, more preferably between 98% and 102%, even more preferably between 99% and 101%, of the number of ribonucleotides in the corresponding antisense ribonucleotide sequence to which it hybridises.
  • the number of ribonucleotides in the sense ribonucleotide sequence is the same as the number of ribonucleotides in the corresponding antisense ribonucleotide sequence.
  • the overall length of the RNA molecule of the invention, produced as a single strand of RNA, after splicing out of any introns but before any processing of the RNA molecule by Dicer enzymes or other RNAses, is typically between 50 and 2000 ribonucleotides, preferably between 60 or 70 and 2000 ribonucleotides, more preferably between 80 or 90 and 2000 ribonucleotides, even more preferably between 100 or 110 and 2000 ribonucleotides.
  • the minimum length of the RNA molecule is 120, 130, 140, 150, 160, 180, or 200 nucleotides, and the maximum length is 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1500 or 2000 ribonucleotides.
  • the maximum length is 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1500 or 2000 ribonucleotides.
  • Production of RNA molecules of such lengths by transcription in vitro or in cells such as bacterial or other microbial cells, preferably S. cerevisiae cells, or in the eukaryotic cell where the target RNA molecule is to be down-regulated, is readily achieved.
  • the present invention provides a chimeric ribonucleic acid (RNA) molecule, comprising a double-stranded RNA (dsRNA) region which comprises a sense ribonucleotide sequence and an antisense ribonucleotide sequence which are capable of hybridising to each other to form the dsRNA region, wherein i) the sense ribonucleotide sequence consists of, covalently linked in 5’ to 3’ order, a first 5’ ribonucleotide, a first RNA sequence and a first 3’ ribonucleotide, ii) the antisense ribonucleotide sequence consists of, covalently linked in 5’ to 3’ order, a second 5’ ribonucleotide, a second RNA sequence and a second 3’ ribonucleotide, iii) the first 5’ ribonucleotide basepairs with the second 3’ ribon
  • the present invention provides a chimeric RNA molecule comprising a first RNA component and a second RNA component which is covalently linked to the first RNA component, wherein the first RNA component comprises a first double-stranded RNA (dsRNA) region, which comprises a first sense ribonucleotide sequence and a first antisense ribonucleotide sequence which are capable of hybridising to each other to form the first dsRNA region, and a first intervening ribonucleotide sequence of at least 4 nucleotides which covalently links the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence, wherein the second RNA component comprises a second sense ribonucleotide sequence, a second antisense ribonucleotide sequence and a second intervening ribonucleotide sequence of at least 4 ribonucleotides which covalently links the second sense ribonucleo
  • the chimeric RNA molecule or at least some of the asRNA molecules, or both are capable of reducing the expression or activity of a target RNA molecule which modulates the timing of plant flowering, or
  • the first antisense ribonucleotide sequence comprises a sequence of at least 20 contiguous ribonucleotides which is at least 50% identical in sequence, preferably at least 90% or 100% identical in sequence, to a region of the complement of the target RNA molecule, or
  • the first 5’ ribonucleotide and first 3’ ribonucleotide of the first RNA component basepair to each other. That basepair is defined herein as the terminal basepair of the dsRNA region formed by self-hybridisation of the first RNA component.
  • the first sense ribonucleotide sequence is linked covalently to the first 5’ ribonucleotide without any intervening nucleotides and the first antisense ribonucleotide sequence is linked covalently to the first 3’ ribonucleotide without any intervening nucleotides
  • the first 5’ ribonucleotide is directly linked to one of the sense sequence and antisense sequence and the first 3’ ribonucleotide is directly linked to the other of the sense sequence and antisense sequence.
  • the RNA molecule comprises one or more or all of (i) a linking ribonucleotide sequence which covalently links the first and second RNA components, (ii) a 5’ extension sequence and (iii) a 3’ extension sequence, wherein the 5’ extension sequence, if present, consists of a sequence of ribonucleotides which is covalently linked to the first RNA component or to the second RNA component, and wherein the 3’ extension sequence, if present, consists of a sequence of ribonucleotides which is covalently linked to the second RNA component or to the first RNA component, respectively.
  • first RNA component and the second RNA component are covalently linked via a linking ribonucleotide sequence.
  • the first RNA component and the second RNA component are directly linked, without any linking ribonucleotide sequence present.
  • the RNA molecule is capable of being made enzymatically by transcription in vitro or in a cell, or both.
  • an RNA molecule of the present invention is expressed in a plant cell i.e. produced in the cell by transcription from one or more nucleic acids encoding the RNA molecule.
  • the one or more nucleic acids encoding the RNA molecule is preferably a DNA molecule, which may be present on a vector in the cell or integrated into the genome of the cell, either the nuclear genome of the cell or in the plastid DNA of the cell.
  • the one or more nucleic acids encoding the RNA molecule may also be an RNA molecule such as a viral vector.
  • the present invention provides an isolated and/or exogenous polynucleotide encoding an RNA molecule of the invention, or a chimeric RNA molecule of the invention.
  • the polynucleotide is a DNA construct.
  • the polynucleotide is operably linked to a promoter capable of directly expression of the RNA molecule in a plant cell.
  • promoters include, but are not limited to an RNA polymerase promoter such as an RNA polymerase III promoter, an RNA polymerase II promoter, or a promoter which functions in vitro.
  • the polynucleotide encodes an RNA precursor molecule comprising an intron in at least one loop sequence which is capable of being spliced out during transcription of the polynucleotide in a plant cell or in vitro.
  • the polynucleotide is a chimeric DNA which comprises in order, a promoter capable of initiating transcription of the RNA molecule in a host cell, operably linked to a DNA sequence which encodes the RNA molecule, preferably a hpRNA, and a transcription termination and/or polyadenylation region.
  • the RNA molecule comprises a hairpin RNA structure which comprises a sense ribonucleotide sequence, a loop sequence and an antisense ribonucleotide sequence, more preferably wherein the sense and antisense ribonucleotide sequences basepair to form a dsRNA region wherein between about 5% and about 40% of the ribonucleotides in the dsRNA region are basepaired in non-canonical basepairs, preferably G:U basepairs.
  • polynucleotides of the invention comprise a nucleotide sequence set forth in SEQ ID NO: 150 or a nucleotide sequence 95% identical thereto. In an embodiment, polynucleotides of the invention comprise a nucleotide sequence set forth in SEQ ID NO: 150.
  • the vector is a viral vector.
  • the vector is a plasmid vector such as a binary vector suitable for use with Agrobacterium tumefaciens.
  • the promoter region of the polynucleotide or vector, which is operably linked to the region which encodes an RNA molecule of the invention has a lower level of methylation when compared to the promoter of a corresponding polynucleotide or vector encoding an RNA molecule which has a corresponding dsRNA region which is fully basepaired with canonical basepairs.
  • the lower level of methylation is less than 50%, less than 40%, less than 30% or less than 20%, when compared to the promoter of the corresponding polynucleotide or vector.
  • the host cell comprises at least two copies of the polynucleotide or vector encoding an RNA molecule of the invention.
  • the level of reduction in the expression and/or activity of the target RNA molecule in the plant cell is at least the same relative to a corresponding plant cell having a single copy of the polynucleotide or vector, and/or ii) the level of reduction in the expression and/or activity of the target RNA molecule in the plant cell is lower when compared to a corresponding cell comprising an RNA molecule which has a corresponding dsRNA region which is fully basepaired with canonical basepairs.
  • the present invention provides a host cell comprising one or more or all of an RNA molecule of the invention, a chimeric RNA molecule of the invention, small RNA molecules (20-24nt in length) produced by processing of the RNA molecule or chimeric RNA molecule, a polynucleotide of the invention, or a vector of the invention.
  • the host cell may be a bacterial cell such as E. coli, a fungal cell such as a yeast cell, for example, S. cerevisiae, or a eukaryotic cell sush as a plant cell.
  • the promoter is heterologous relative to the polynucleotide.
  • the polynucleotide encoding the RNA molecule may be a chimeric or recombinant polynucleotide, or an isolated and/or exogenous polynucleotide.
  • the promoter can function in vitro, for example a bacteriophage promoter such as a T7 RNA polymerase promoter or SP6 RNA polymerase promoter.
  • the promoter is an RNA polymerase III promoter such as a U6 promoter or an HI promoter.
  • the promoter is an RNA polymerase II promoter, which may be a constitutive promoter, a tissue-specific promoter, a developmental ⁇ regulated promoter or an inducible promoter.
  • the polynucleotide encodes an RNA precursor molecule comprising an intron in at least one loop sequence which is capable of being spliced out during or after transcription of the polynucleotide in a host cell.
  • the host cell is a plant cell.
  • the promoter region of the polynucleotide has a lower level of methylation, such as less than about 50%, less than about 40%, less than about 30% or less than about 20%, when compared to the promoter of a corresponding polynucleotide encoding an RNA molecule which has a corresponding dsRNA region which is fully basepaired with canonical basepairs.
  • the host cell is a plant cell comprising the chimeric RNA molecule or small RNA molecules produced by processing of the chimeric RNA molecule, or both, wherein the chimeric RNA molecule comprises, in 5’ to 3’ order, the first sense ribonucleotide sequence, the first linking ribonucleotide sequence which comprises a loop sequence, and the first antisense ribonucleotide sequence.
  • the plant cell may be from Arabidopsis, com, canola, cotton, soybean, alfalfa, lettuce, wheat, barley, rice, legume, Medicago truncatula, sugarbeet or rye.
  • the plant cell may be from Arabidopsis, com, canola, cotton, soybean, wheat, barley, rice, legume, Medicago truncatula, sugarbeet or rye.
  • the host cell comprises at least two copies of the polynucleotide, and wherein i) the level of reduction in the expression or activity of the target RNA molecule in a plant cell is at least the same when compared to if the cell had a single copy of the polynucleotide, and/or ii) the level of reduction in the expression or activity of the target RNA molecule in a plant cell is lower when compared to a corresponding cell comprising an RNA molecule which has a corresponding dsRNA region which is fully basepaired with canonical basepairs.
  • the cell encodes and/or comprises the chimeric RNA molecule of the invention and the level of sense ribonucleotide sequence in the cell is less than 50 to 99% the level of the antisense ribonucleotide.
  • the RNA molecule is expressed in a eukaryotic cell i.e. produced by transcription in the cell.
  • a greater proportion of dsRNA molecules are formed by processing of the RNA molecule that are 22 and/or 20 ribonucleotides in length when compared to processing of an analogous RNA molecule which has a corresponding dsRNA region which is fully basepaired with canonical basepairs.
  • RNA molecules of these embodiments are more readily processed to provide 22- and/or 20-ribonucleotide short antisense RNAs than the analogous RNA molecule whose dsRNA region is fully basepaired with canonical basepairs, as a proportion of the total number of 20-24 nucleotide asRNAs produced from the RNA molecule.
  • a lesser proportion of dsRNA molecules are formed by processing of the RNA molecule that are 23 and/or 21 ribonucleotides in length when compared to processing of an analogous RNA molecule which has a corresponding dsRNA region which is fully basepaired with canonical basepairs.
  • the RNA molecules of these embodiments are less readily processed to provide 23- and/or 21 -ribonucleotide short antisense RNAs than the analogous RNA molecule whose dsRNA region is fully basepaired with canonical basepairs, as a proportion of the total number of 20-24 nucleotide asRNAs produced from the RNA molecule.
  • at least 50% of the RNA transcripts produced in the cell by transcription from the genetic construct are not processed by Dicer.
  • RNA molecules that are formed by processing of the RNA molecule have more than one phosphate covalently attached at the 5’ terminus when compared to processing of an analogous RNA molecule which has a corresponding dsRNA region which is fully basepaired with canonical basepairs. That is, a greater proportion of the short antisense RNA molecules have an altered charge which can be observed as a mobility shift of the molecules in gel electrophoresis experiments.
  • the present invention provides a plant comprising one or more or all of an RNA molecule of the invention, a chimeric RNA molecule of the invention, small RNA molecules (20-24nt in length) produced by processing of the RNA molecule or chimeric RNA molecule, a polynucleotide of the invention, a vector of the invention, or a host cell of the invention which is a plant cell.
  • the plant is transgenic insofar as it comprises a polynucleotide of the invention.
  • the polynucleotide is stably integrated into the genome of the plant.
  • the invention also includes plant parts, and products obtained therefrom, comprising the RNA molecule or small RNA molecules (20-24nt in length) produced by processing of the chimeric RNA molecule, or both, and/or the polynucleotide or vector of the invention, for example to seeds, crops, harvested products and post-harvest products produced therefrom.
  • the present invention provides a method of producing an RNA molecule of the invention, or a chimeric RNA molecule of the invention, the method comprising expressing the polynucleotide of the invention in a host cell or cell- free expression system.
  • the method further comprises at least partially purifying the RNA molecule.
  • the present invention provides a method of producing the plant of the invention, the method comprising introducing the polynucleotide of the invention into a plant cell so that it is stably integrated into the genome of the cell, and generating the plant from the cell.
  • the present invention provides a method of producing a cell or plant, the method comprising introducing a polynucleotide or vector or RNA molecule of the invention into a plant cell, preferably so that the polynucleotide or vector or part thereof encoding the RNA molecule is stably integrated into the genome of the plant cell.
  • the plant is generated from the cell or a progeny cell, for example by regenerating a transgenic plant and optionally producing progeny plants therefrom.
  • the plant is generated by introducing the cell or one or more progeny cells into the plant.
  • the polynucleotide or vector may be introduced into the cell without integration of the polynucleotide or vector into the genome, for example to produce the RNA molecule transiently in the plant cell or plant.
  • the plant is resistant to a pest or pathogen, e.g. a plant pest or pathogen, preferably an insect pest or fungal pathogen.
  • the method comprises a step of testing one or more plants, comprising the polynucleotide or vector or RNA molecule of the disclosure for modulation of flowering.
  • the plants that are tested may be progeny from the plant, into which the polynucleotide or vector or RNA molecule of the disclosure was first introduced, and therefore the method may comprise a step of obtaining such progeny.
  • the method may further comprise a step of identifying and/or selecting the plant with desired time to flowering such as early flowering. For example, multiple plants, which each comprise the polynucleotide or vector or RNA molecule of the invention may be tested to identify those with desired time to flowering, and progeny obtained from the identified plant(s).
  • the present invention provides an extract of a host cell of the invention, wherein the extract comprises the RNA molecule of the invention, a chimeric RNA molecule of the invention, small RNA molecules (20-24nt in length) produced by processing of the RNA molecule or chimeric RNA molecule, or both, and/or the polynucleotide of the invention.
  • the present invention provides a composition comprising one or more of an RNA molecule of the invention, a chimeric RNA molecule of the invention, small RNA molecules (20-24nt in length) produced by processing of the RNA molecule or chimeric RNA molecule, a polynucleotide of the invention, a vector of the invention, a host cell of the invention, or an extract of the invention, and one or more suitable carriers.
  • the composition is suitable for application to a field, e.g. as topical spray.
  • the field comprises plants.
  • the composition is suitable for application to a crop, for example by spraying on crop plants in a field.
  • the composition further comprises at least one compound which enhances the stability of the RNA molecule, chimeric RNA molecule or polynucleotide and/or which assists in the RNA molecule, chimeric RNA molecule or polynucleotide being taken up by a cell of a plant.
  • the compound is a transfection promoting agent.
  • the present invention provides a method for down-regulating the level and/or activity of a target RNA molecule which modulates plant flowering in a plant, the method comprising delivering to the plant one or more of an RNA molecule of the invention, a chimeric RNA molecule of the invention, small RNA molecules (20- 24nt in length) produced by processing of the RNA molecule or chimeric RNA molecule, a polynucleotide of the invention, a vector of the invention, a host cell of the invention, an extract of the invention, or a composition of the invention.
  • delivering may be via contacting, exposing, transforming or otherwise introducing an RNA molecule or chimeric RNA molecule disclosed herein or a mixture thereof, or small RNA molecules (20-24nt in length) produced by processing of the RNA molecule or chimeric RNA molecule or the polynucleotide or vector of the invention to the plant cell or plant.
  • the introduction may be enhanced by use of an agent that increases the uptake of the RNA molecule(s), polynucleotides or vectors of the invention, for example with the aid of transfection promoting agents, DNA- or RNA-binding polypeptides, or may be done without adding such agents, for example by planting seed which is transgenic for a polynucleotide or vector of the invention and allowing the seed to grow into a transgenic plant which expresses the RNA molecules of the invention.
  • the target RNA molecule encodes a protein.
  • the method reduces the level and/or activity of more than one target RNA molecule, the target RNA molecules being different, for example two or more target RNAs are reduced in level and/or activity which are related in sequence such as from a gene family.
  • the chimeric RNA molecule or small RNA molecules produced by processing of the chimeric RNA molecule, or both are contacted with the cell or organism, preferably a plant cell or plant by topical application to the cell or organism, or provided in a feed for the organism.
  • the target RNA molecule encodes a protein.
  • one or more of the target RNAs do not encode a protein, such as a rRNA, tRNA, snoRNA or miRNA.
  • the chimeric RNA molecule, or small RNA molecules produced by processing of the chimeric RNA molecule, or both are contacted with the cell or plant by topical application to the cell or plant.
  • the present disclosure encompasses a method of promoting flowering time of a plant, the method comprising expressing a polynucleotide heterologous to said plant, wherein said polynucleotide heterologous to said plant is a polynucleotide of the invention such as an RNA molecule of the invention, wherein expression of said polynucleotide in said plant directs early flowering.
  • the present invention provides a method of modulating the flowering time of a plant, or a plant produced from a seed, the method comprising contacting the plant or seed with a composition comprising an RNA molecule which comprises at least one double stranded RNA region, and/or a polynucleotide/ s) encoding the RNA molecule, wherein the at least one double stranded RNA region comprises an antisense ribonucleotide sequence which is capable of hybridising to a region of a target RNA molecule which modulates the timing of plant flowering.
  • the composition is an aqueous composition.
  • the composition comprises at least one compound which enhances the stability of the RNA molecule and/or which assists in the RNA molecule being taken up by a cell of a plant.
  • the compound is a transfection promoting agent.
  • the method comprises soaking the seed in the composition.
  • the plant is a seedling, and the method comprises soaking at least a part of the seedling in the composition.
  • at least a part, or all, of the cotyledon(s) and/or the hypocotyl are soaked in the composition.
  • the plant is in a field and the method comprising spraying the composition on at least a part of the plant.
  • the RNA molecule can have any suitable structure for gene silencing. Examples include, but are not limited to, hairpin RNA, a microRNA, a siRNA or an ledRNA.
  • the RNA molecule of the above aspect can be a chimeric RNA molecule such as described herein.
  • the nature in which flowering time is modulated will depend on the taget RNA molecule.
  • the plant has an early flowering time when compared to a control plant that has not been applied with the composition.
  • the plant has a late flowering time when compared to a control plant that has not been applied with the composition. Examples of target RNA molecules to be targeted to induce early or late falowering are discussed herein.
  • the RNA molecule is complexed with a non-RNA molecule such as DNA, a protein or a polymer.
  • the complex comprises the RNA molecule conjugated to the non-RNA molecule such as by a covalent bond.
  • the composition is topically applied to the plant or seed.
  • the polynucleotide is present in the composition in a cell and/or a vector.
  • the present invention provideds a kit comprising one or more of an RNA molecule of the invention, a chimeric RNA molecule of the invention, a polynucleotide of the invention, a vector of the invention, a host cell of the invention, an extract of the invention, or a composition of the invention.
  • the kit may further comprise instructions for use of the kit.
  • the present invention provides a process for producing dsRNA molecules, the process comprising a) culturing S. cerevisiae expressing one or more polynucleotides encoding one or more dsRNA molecules, and b) harvesting the S. cerevisiae producing the dsRNA molecules, or the dsRNA molecules from the S. cerevisiae , wherein the S. cerevisiae are cultured in a volume of at least 1 litre.
  • the dsRNA can have any structure, such as an hairpin RNA (for example shRNA), a miRNA or a dsRNA of the invention.
  • the S. cerevisiae are cultured in a volume of at least 10 litres, at least 100 litres, at least 1,000 litres, at least 10,000 litres or at least 100,000 litres.
  • the process produces at least 0.1, at least 0.5 or at least 1 g/litre of an RNA molecule of the invention.
  • the S. cerevisiae produced using the process, or dsRNA molecules isolated therefrom can be used in methods described herein such as, but not limited to, a method for reducing or down-regulating the level and/or activity of a target RNA molecule in a cell or plant.
  • composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.
  • This ledRNA molecule comprises a sense sequence which can be considered to be two adjacent sense sequences, covalently linked without an intervening spacer sequence and having identity to the target RNA, an antisense sequence which is complementary to the sense sequence and which is divided into two regions, a 5’ region and a 3’ region, and two loops that separate the sense from the antisense sequences.
  • This ledRNA molecule comprises an antisense sequence which can be considered to be two adjacent antisense sequences, covalently linked without an intervening spacer sequence and having identity to the complement of a target RNA, a sense sequence which is complementary to the antisense sequence and which is divided into two regions, and two loops that separate the sense from the antisense sequences.
  • the RNA molecule produced by transcription for example by in vitro transcription from a promoter such as a T7 or Sp6 promoter, self-anneals by basepairing between the complementary sense and antisense sequences to form a double-stranded region with a loop at each end and having a “nick” in either the antisense or sense sequence.
  • Additional sequences may be linked to the 5’ and/or 3’ ends as 5’- or 3 ’-extensions.
  • Figure 7 Alignment of the nucleotide sequences of a region of the GUS target gene (SEQ ID NO: 14) and the sense sequence of the hpGUS[G:U] construct (nucleotides 9 to 208 of SEQ ID NO: 11). 52 cytidine (C) nucleotides were substituted with thymidine (T) nucleotides. conserveed nucleotides are asterisked, substituted C’s are not asterisked.
  • Figure 8 Alignment of the nucleotide sequences of a region of the GUS target gene (SEQ ID NO:14) and the sense sequence of the hpGUS[l:4] construct (nucleotides 9 to 208 of SEQ ID NO: 12). Every 4th nucleotide in hpGUS[l:4] was substituted relative to the corresponding wild-type sense sequence, whereby for every 4th nucleotide, C was changed to G, G was changed to C, A was changed to T, and T was changed to A. conserveed nucleotides are asterisked, substituted G’s and C’s are not asterisked, substituted A’s and T’s are shown with semi-colons.
  • Figure 9 Alignment of the nucleotide sequences of a region of the GUS target gene (SEQ ID NO: 14) and the sense sequence of the hpGUS[2:10] construct (nucleotides 9 to 208 of SEQ ID NO: 13). Every 9th and 10th nucleotide in each block of 10 nucleotides in hpGUS[2:10] was substituted relative to the corresponding wild-type sense sequence, whereby for every 9th and 10th nucleotide, C was changed to G, G was changed to C, A was changed to T and T was changed to A.
  • FIG. 10 Schematic diagram showing structures of the genetic constructs encoding modified hairpin RNAs targeting GUS mRNA.
  • FIG. 11 Schematic diagram of vector pWBPPGH used to transform tobacco plants, providing a GUS target gene.
  • the T-DNA extends from the right border (RB) to the left border (LB) of the vector.
  • the selectable marker gene on the T-DNA is the 35S- HPT-tml’ gene encoding hygromycin resistance.
  • FIG. 12 GUS activity in plants transformed with constructs encoding modified hairpin RNAs for reducing expression of a GUS target gene.
  • No hp control PPGH11 and PPGH24 plants with no hpGUS constructs.
  • the number of plants showing less than 10% GUS activity compared to the corresponding control PPGH11 or PPGH24 plants and the percentage of such plants relative to the number of plants tested are given in brackets.
  • FIG. 13 Average GUS activity of all transgenic plants: 59 plants for hpGUS [wt], 74 for hpGUS [G:U], 33 for hpGUS [1:4] and 41 for hpGUS [2: 10]
  • B Average GUS activity of all silenced plants (32 for hpGUS [wt], 71 for hpGUS [G:U], 33 for hpGUS [1:4] and 28 for hpGUS [2: 10]
  • Figure 15 Autoradiograph of a Southern blot of DNA from 16 plants transformed with the hpGUS [G:U] construct. DNAs were digested with Hindi! prior to gel electrophoresis and probed with an OCS-T probe. Lane 1: size markers (Hindlll- digested lambda DNA); Lanes 2 and 3, DNA from parental plants PPGH11 and PPGH24; Lanes 4-19: DNAs from 16 different transgenic plants.
  • Figure 16 Autoradiogram of a Northern blot hybridisation experiment to detect sense (upper panel) and antisense (lower panel) sRNAs derived from hairpin RNAs expressed in transgenic tobacco plants.
  • Lanes 1 and 2 contained RNA obtained from the parental plants PPGH11 and PPGH24 lacking the hpGUS constructs.
  • Lanes 3-11 contained RNA from hpGUS [wt] plants and lanes 12-20 contained RNA from hpGUS [G:U] plants.
  • Figure 17 Autoradiograph of a Northern blot hybridisation to detect antisense sRNAs from transgenic plants. Lanes 1-10 were from hpGUS[wt] plants, lanes 11-19 were from hpGUS[G:U] plants. The antisense sRNAs have mobility corresponding to 20- 24nt in length. The blot was reprobed with antisense to U6 RNA as a lane-loading control.
  • FIG. 18 Autoradiograph of a repeat Northern blot hybridisation to detect antisense sRNAs from transgenic plants
  • Figure 19 DNA methylation analysis of the junction region of the 35S promoter and sense GUS region in hpGUS constructs in transgenic plants. The junction fragments were PCR-amplified either with (+) or without (-) prior treatment of plant DNA with McrBC enzyme.
  • FIG. 20 DNA methylation analysis of the 35S promoter region in hpGUS constructs in transgenic plants.
  • the 35S fragments were PCR-amplified either with (+) or without (-) prior treatment of plant DNA with McrBC enzyme.
  • Figure 22 Alignment of the sense sequence (upper sequence, nucleotides 17 to 216 of SEQ ID NO:22) of the hpEIN2[G:U] construct and the nucleotide sequence (lower sequence, SEQ ID NO:27) of a region of the cDNA corresponding to the A. thaliana EIN2 target gene.
  • the sense sequence was made by replacing 43 cytidine (C) nucleotides in the wild-type sequence with thymidine (T) nucleotides. conserveed nucleotides are asterisked, substituted C’s are not asterisked.
  • Figure 23 Alignment of the sense sequence (upper sequence, nucleotides 13 to 212 of SEQ ID NO:24) of the hpCHS[G:U] construct with the nucleotide sequence of a region of the cDNA corresponding to the A. thaliana CHS target gene (SEQ ID NO:28, lower sequence).
  • the sense sequence was made by replacing 65 cytidine (C) nucleotides in the wild-type sequence with thymidine (T) nucleotides. conserveed nucleotides are asterisked, substituted C’s are not asterisked.
  • Figure 24 Alignment of the antisense sequence (upper sequence, nucleotides 8 to 207 of SEQ ID NO:25) of the hpEIN2[G:U/U:G] construct and the nucleotide sequence (lower sequence, SEQ ID NO:29) of a region of the complement of the A. thaliana EIN2 target gene and the.
  • the antisense sequence was made by replacing 49 cytidine
  • C nucleotides in the wild-type sequence with thymidine (T) nucleotides. conserveed nucleotides are asterisked, substituted C’s are not asterisked.
  • Figure 25 Alignment of the antisense sequence (upper sequence, nucleotides 13 to 212 of SEQ ID NO:26) of the hpCHS[G:U/U:G] construct and the nucleotide sequence (lower sequence, SEQ ID NO:30) of a region of the complement of the A. thaliana CHS target gene.
  • the antisense sequence was made by replacing 49 cytidine (C) nucleotides in the wild-type sequence with thymidine (T) nucleotides. conserveed nucleotides are asterisked, substituted C’s are not asterisked.
  • FIG. 26 Schematic diagrams of the ethylene insensitive 2 (EIN2) and chalcone synthase (CHS) hpRNA constructs.
  • 35S CaMV 35S promoter; EIN2 and CHS regions are show either as wild-type sequence (wt) or the G:U modified sequence (G:U).
  • the arrows indicate the orientation of the DNA fragments - right to left arrows indicate the antisense sequences. Restriction enzyme sites are also shown.
  • FIG. 27 Hypocotyl lengths of transgenic A. thaliana seedlings in the EIN2 assay, containing either the hpEIN2[wt] or hpEIN2[G:U]
  • FIG. 28 qRT-PCR for CHS mRNA in transgenic A. thaliana transgenic for the hpCHS[wt] or hpCHS[G:U] constructs, normalised to the levels of Actin2 RNA.
  • Col-0 is the wild-type (nontransgenic) A. thaliana.
  • FIG. 29 Autoradiograph of Northern blot hybridisation of RNA from plants transformed with hpEIN2[wt] or hpEIN2[G:U]. Upper panel shows the hypocotyl length for the lines. The autoradiograph shows Northern blot probed with an EIN2 sense probe to detect antisense sRNAs. The same blot was re-probed with a U6 RNA probe as a loading control (U6 RNA).
  • Figure 30 DNA methylation analysis of 35S promoter and 35S-sense EIN2 sequences in genomic DNA of transgenic A. thaliana plants.
  • Figure 31 Levels of DNA methylation in the promoter and 5’ region of hairpin RNA constructs.
  • Figure 34 ledRNA and hpRNA with G:U gene silencing in CHO and Vero cells at 72 hrs.
  • Figure 35 Dumbbell plasmids tested in Hela cells at 48 hrs.
  • FIG 37 Reduced aphid performance following feeding from artificial diet supplemented with ledRNA for down-regulating expression of the MpC002 or MpRack-1 genes in green peach aphid.
  • Upper panel (A) the average number of nymphs per adult aphid after a ten day period with 100 pi of 50ng/pl ledRNA.
  • Lower panel (B) percentage of aphids surviving over a five day time course after feeding on 100 m ⁇ containing 200ng/pl ledRNA of MpC002, MpRack-1 or the control ledGFP.
  • Figure 38 Northern blot hybridization to detect ledGUS and hpGUS RNA using full- length sense GUS transcript as probe. “+” at the bottom indicates high GUS expression; indicates low/no GUS expression i.e. strong GUS silencing.
  • Figure 39 Northern blot hybridization to detect long hpEIN2 and ledEIN2 RNA (upper panel) and siRNAs derived from the two constructs (lower panel).
  • Figure 40 Schematic representation of stem-loop structures of transcripts expressed from GUS hpRNA constructs. The transcripts have complementary sense and antisense sequences which basepair to form GUS sequence- specific dsRNA stems, with the indicated lengths in basepairs (bp) for the stems, and the number of nucleotides (nt) in the loops.
  • the GFP hpRNA constructs encoded transcripts that formed a GFP-specific dsRNA stem with completely canonical basepairing (GFPhp[WT] or a dsRNA stem having about 25% of basepairs as G:U base-pairs (GFPhp[G:U], with a loop derived from a region of GUS coding sequence.
  • the loop sequences for the GFPhp transcripts each comprised two sequences that were complementary to miR165/miR166 and therefore provide binding sites for these miRNAs.
  • FIG. 41 Northern blot hybridisation analysis showing that transgenes encoding hpRNAs generate distinct fragments of the loop sequence when expressed in plant cells.
  • GUS GUS target gene
  • CMV2b cucumber mosaic virus 2b RNA silencing suppressor
  • FIG 43 Transgenic S. cerevisiae expressing a GUShpllOO construct showed a single RNA molecular species corresponding to the full length hairpin RNA transcript.
  • the lower panel shows the Northern blot hybridisation of RNA samples from the transgenic S. cerevisiae.
  • FIG. 44 GUShpllOO transcript expressed in S. cerevisiae remains full-length and does not form circular loop RNA.
  • hpRNA loops may be used as an effective sequence-specific repressor of miRNAs.
  • A The GFPhp[G:U] construct induced strong miR165/166 suppression phenotypes in transgenic Arabidopsis plants.
  • B Northern blot hybridization to determine the abundance of GFPhp transcript in RNA from transgenic Arabidopsis plants.
  • C RT-qPCR analysis of circular RNA of the GFPhp loop.
  • FIG. 46 Treatment of wheat seedlings with ledTaVRN2 reduced the requirement of winter wheat for vernalisation prior to initiation of flowering.
  • Chinese Spring is a spring type wheat that does not require vernalisation, used as a control.
  • FIG 47 Treatment of the winter wheat variety CSIRO W7 with ledTaVRN2 induced earlier flowering compared to ledGFP, mock and no treatment controls.
  • Early flowering parental genotypes Sunstate A (SSA) and Sunstate B (SSB) lacked a vernalisation response and were included as controls.
  • SEQ ID NO:2 Ribonucleotide sequence of GUS ledRNA.
  • SEQ ID NO:4 Nucleotide sequence encoding GFP ledRNA.
  • SEQ ID NO: 10 Nucleotide sequence used to provide the GUS sense region for constructs encoding hairpin RNA molecules targeting the GUS mRNA.
  • SEQ ID NO: 11 Nucleotide sequence used to provide the GUS sense region for the construct encoding the hairpin RNA molecule hpGUS[G:U].
  • SEQ ID NO: 12 Nucleotide sequence used to provide the GUS sense region for constructs encoding the hairpin RNA molecule hpGUS[l:4].
  • SEQ ID NO: 13 Nucleotide sequence used to provide the GUS sense region for constructs encoding the hairpin RNA molecule hpGUS[2:10].
  • SEQ ID NO: 14 Nucleotide sequence of nucleotides 781-1020 of the protein coding region of the GUS gene.
  • SEQ ID NO: 15 Ribonucleotide sequence of the hairpin structure (including its loop) of the hpGUS[wt] RNA.
  • SEQ ID NO: 16 Ribonucleotide of the hairpin structure (including its loop) of the hpGUS[G:U] RNA.
  • SEQ ID NO: 17 Ribonucleotide of the hairpin structure (including its loop) of the hpGUS[l:4] RNA.
  • SEQ ID NO: 18 Ribonucleotide of the hairpin structure (including its loop) of the hpGUS[2:10] RNA.
  • SEQ ID NO: 19 Nucleotide sequence of the cDNA corresponding to the A. thaliana EIN2 gene, Accession No. NM_120406.
  • SEQ ID NO:20 Nucleotide sequence of the cDNA corresponding to A. thaliana CHS gene, Accession No. NM_121396, 1703nt.
  • SEQ ID NO:21 Nucleotide sequence of a DNA fragment comprising a 200nt sense sequence from the cDNA corresponding to the A. thaliana EIN2 gene flanked by restriction enzyme sites.
  • SEQ ID NO:22 Nucleotide sequence of a DNA fragment comprising the 200nt sense sequence of EIN2 as for SEQ ID NO:21 except that 43 C’s were replaced with T’s, used in constructing hpEIN2[G:U].
  • SEQ ID NO:23 Nucleotide sequence of a DNA fragment comprising a 200nt sense sequence from the cDNA corresponding to A. thaliana CHS gene flanked by restriction enzyme sites.
  • SEQ ID NO:24 Nucleotide sequence of a DNA fragment comprising the 200nt sense sequence of CHS as for SEQ ID NO:23 except that 65 C’s were replaced with T’s, used in constructing hpCHS[G:U].
  • SEQ ID NO:25 Nucleotide sequence of a DNA fragment comprising the 200nt antisense sequence of EIN2 with 50 C’s replaced with T’s, used in constructing hpEIN2[G:U/U:G]
  • SEQ ID NO:26 Nucleotide sequence of a DNA fragment comprising the 200nt antisense sequence of CHS with 49 C’s replaced with T’s, used in constructing hpCHS[G:U/U:G]
  • SEQ ID NO:27 Nucleotide sequence of nucleotides 601-900 of the cDNA corresponding to the EIN2 gene from A. thaliana (Accession No. NM_120406).
  • SEQ ID NO:28 Nucleotide sequence of nucleotides 813-1112 of the cDNA corresponding to the CHS gene from A. thaliana (Accession No. NM_121396).
  • SEQ ID NO:29 Nucleotide sequence of the complement of nucleotides 652-891 of the cDNA corresponding to the EIN2 gene from A. thaliana (Accession No. NM_ 120406).
  • SEQ ID NO:30 Nucleotide sequence of the complement of nucleotides 804-1103 of the cDNA corresponding to the CHS gene from A. thaliana.
  • Target region nucleotides 675-1174 (500 nucleotides) SEQ ID NO:32 - FANCM I protein coding region of a cDNA of Brassica napus.
  • SEQ ID NO:33 Nucleotide sequence encoding hpFANCM-At[wt] targeting the FANCM I protein coding region of A. thaliana.
  • FANCM sense sequence nucleotides 38-537; loop sequence, nucleotides 538-1306; FANCM antisense sequence, nucleotides 1307-1806.
  • SEQ ID NO:34 Nucleotide sequence encoding hpFANCM-At[G:U] targeting the FANCM I protein coding region of A. thaliana.
  • FANCM sense sequence nucleotides 38-537; loop sequence, nucleotides 538-1306; FANCM antisense sequence, nucleotides 1307-1806.
  • SEQ ID NO:35 Nucleotide sequence encoding hpFANCM-Bn[wt] targeting the FANCM I protein coding region of B. napus.
  • FANCM sense sequence nucleotides 34- 533; loop sequence, nucleotides 534-1300; FANCM antisense sequence, nucleotides 1301-1800.
  • SEQ ID NO:36 Nucleotide sequence encoding hpFANCM-Bn[G:U] targeting the FANCM I protein coding region of B. napus.
  • FANCM sense sequence nucleotides 34- 533; loop sequence, nucleotides 534-1300; FANCM antisense sequence, nucleotides 1301-1800.
  • SEQ ID NO:37 Nucleotide sequence of the protein coding region of the cDNA corresponding to the B. napus DDM1 gene; Accession No. XR_001278527.
  • SEQ ID NO:38 Nucleotide sequence of DNA encoding hpDDMl-Bn[wt] targeting the DDM1 protein coding region of B. napus.
  • SEQ ID NO:39 Nucleotide sequence encoding hpDDMl-Bn[G:U] targeting the DDM1 protein coding region of B. napus.
  • DDM1 sense sequence nucleotides 35-536; loop sequence, nucleotides 537-1304; DDM1 antisense sequence, nucleotides 1305- 1805.
  • SEQ ID NO:41 Nucleotide sequence of the coding region of hpEGFP[wt], with the order antisense/ loop/ sense with respect to the promoter.
  • SEQ ID NO:42 Nucleotide sequence of the coding region of hpEGFP[G:U] which has 157 C to T substitutions in the EGFP sense sequence.
  • SEQ ID NO:43 Nucleotide sequence of the coding region of ledEGFP[wt] which has no C to T substitutions in the EGFP sense sequence.
  • SEQ ID NO:44 Nucleotide sequence of the coding region of ledEGFP[G:U] which has 162 C to T substitutions in the EGFP sense sequence.
  • SEQ ID NO:45 Nucleotide sequence used to provide the GUS sense region for the construct encoding the hairpin RNA molecule hpGUS[G:U] without flanking restriction enzyme sites.
  • SEQ ID NO:46 Nucleotide sequence used to provide the GUS sense region for constructs encoding the hairpin RNA molecule hpGUS[l:4] without flanking restriction enzyme sites.
  • SEQ ID NO:47 Nucleotide sequence used to provide the GUS sense region for constructs encoding the hairpin RNA molecule hpGUS[2:10] without flanking restriction enzyme sites.
  • SEQ ID NO:48 Nucleotide sequence of a DNA fragment comprising the 200nt sense sequence of EIN2 as for SEQ ID NO:21 except that 43 C’s were replaced with T’s, used in constructing hpEIN2[G:U] without flanking sequences.
  • SEQ ID NO:49 Nucleotide sequence of a DNA fragment comprising the 200nt sense sequence of CHS as for SEQ ID NO:23 except that 65 C’s were replaced with T’s, used in constructing hpCHS[G:U] without flanking sequences.
  • SEQ ID NO:50 Nucleotide sequence of a DNA fragment comprising the 200nt antisense sequence of EIN2 with 50 C’s replaced with T’s, used in constructing hpEIN2[G:U/U:G] without flanking sequences
  • SEQ ID NO:51 Nucleotide sequence of a DNA fragment comprising the 200nt antisense sequence of CHS with 49 C’s replaced with T’s, used in constructing hpCHS[G:U/U:G] without flanking sequences.
  • SEQ ID NO:52 Oligonucleotide primer used for amplifying the 200 bp GUS sense sequence (GUS-WT-F)
  • SEQ ID NO:54 Oligonucleotide primer (forward) used for producing the hpGUS[G:U] fragment with every C replaced with T (GUS-GU-F)
  • SEQ ID NO:56 Oligonucleotide primer (forward) used for producing the hpGUS[l:4] fragment with every 4th nucleotide substituted (GUS-4M-F)
  • SEQ ID NO:58 Oligonucleotide primer (forward) used for producing the hpGUS[2:10] fragment with every 9th and 10th nucleotide substituted (GUS-10M-F)
  • SEQ ID NO:59 Oligonucleotide primer (reverse) used for producing the hpGUS[2:10] fragment with every 9th and 10th nucleotide substituted (GUS-10M-R)
  • SEQ ID NO:60 Nucleotide sequence encoding forward primer (35S-F3)
  • SEQ ID NO:62 Nucleotide sequence encoding forward primer (GUSgu-R2)
  • SEQ ID NO:68 Oligonucleotide primer used for amplifying the wild-type 200 bp CHS sense sequence (CHSwt-F)
  • SEQ ID NO:70 Oligonucleotide primer (forward) used for producing the hpEIN2[G:U] fragment, with every C replaced with T (EIN2gu-F)
  • SEQ ID NO:72 Oligonucleotide primer (forward) used for producing the hpCHS[G:U] fragment, with every C replaced with T (CHSgu-F)
  • SEQ ID NO:74 Oligonucleotide primer (forward) used for producing the hpEIN2[G:U/U:G] fragment, with every C replaced with T (asEIN2gu-F)
  • SEQ ID NO:76 Oligonucleotide primer (forward) used for producing the hpCHS[G:U/U:G] fragment, with every C replaced with T (asCHSgu-F)
  • SEQ ID NO:81 Nucleotide sequence encoding reverse primer (Actin2-Rev)
  • SEQ ID NO:82 Nucleotide sequence encoding forward primer (Top-35S-F2)
  • SEQ ID NO:86 Ribonucleotide sequence of sense si22 SEQ ID NO:87 - Ribonucleotide sequence of antisense si22 SEQ ID NO:88 - Ribonucleotide sequence of forward primer SEQ ID NO: 89 - Ribonucleotide sequence of reverse primer SEQ ID NO:90 - Ribonucleotide sequence of forward primer SEQ ID NO:91 - Ribonucleotide sequence of reverse primer SEQ ID NO:92 - Possible modifications of dsRNA molecules
  • SEQ ID NO:93 Nucleotide sequence of a cDNA corresponding to the Brassica napus DDM1 gene (Accession No. XR_001278527).
  • SEQ ID NO:94 Nucleotide sequence of a chimeric DNA encoding a hairpin RNAi (hpRNA) construct targeting a DDM1 gene of B. napus.
  • SEQ ID NO:95 Nucleotide sequence of a chimeric DNA encoding a hairpin RNAi (hpRNA) construct with G:U basepairs, targeting a DDM1 gene of B. napus.
  • SEQ ID NO:96 Nucleotide sequence of a chimeric DNA encoding a ledRNA construct, targeting a DDM1 gene of B. napus.
  • SEQ ID NO:97 Nucleotide sequence of cDNA corresponding to A. thaliana FANCM gene (Accession No. NM_001333162).
  • SEQ ID NO:98 Nucleotide sequence of a chimeric DNA encoding a hairpin RNAi (hpRNA) construct targeting a FANCM gene of A. thaliana.
  • SEQ ID NO:99 Nucleotide sequence of a chimeric DNA encoding a hairpin RNAi (hpRNA) construct with G:U basepairs, targeting a FANCM gene of A. thaliana.
  • SEQ ID NO: 100 Nucleotide sequence of a chimeric DNA encoding a ledRNA construct, targeting a FANCM gene of A. thaliana.
  • SEQ ID NO: 101 Nucleotide sequence of cDNA corresponding to B. napus FANCM gene (Accession No. XM_022719486.1).
  • SEQ ID NO: 102 Nucleotide sequence of a chimeric DNA encoding a hairpin RNAi (hpRNA) construct targeting a FANCM gene of B. napus.
  • SEQ ID NO: 103 Nucleotide sequence of a chimeric DNA encoding a hairpin RNAi (hpRNA) construct with G:U basepairs, targeting a FANCM gene of B. napus.
  • SEQ ID NO: 104 Nucleotide sequence of a chimeric DNA encoding a ledRNA construct, targeting a FANCM gene of B. napus.
  • SEQ ID NO: 105 Nucleotide sequence of the protein coding region of the cDNA corresponding to the Nicotiana benthamiana TOR gene.
  • SEQ ID NO: 106 Nucleotide sequence of a chimeric DNA encoding a ledRNA construct targeting a TOR gene of N. benthamiana.
  • SEQ ID NO: 107 Nucleotide sequence of the protein coding region of the cDNA corresponding to the acetolactate synthase ( ALS ) gene of barley, Hordeum vulgare (Accession No. LT601589).
  • SEQ ID NO: 108 Nucleotide sequence of a chimeric DNA encoding a ledRNA targeting the ALS gene of barley ( H . vulgare).
  • SEQ ID NO: 109 Nucleotide sequence of the protein coding region of the cDNA corresponding to the HvNCEDl gene of barley Hordeum vulgare (Accession No. AK361999).
  • SEQ ID NO: 110 Nucleotide sequence the protein coding region of the cDNA corresponding to the HvNCED2 gene of barley Hordeum vulgare (Accession No. DQ145931).
  • SEQ ID NO:lll Nucleotide sequence of a chimeric DNA encoding a ledRNA construct targeting the NCED1 genes of barley Hordeum vulgare and wheat Triticum aestivum.
  • SEQ ID NO: 112 Nucleotide sequence of a chimeric DNA encoding a ledRNA construct targeting the NCED2 genes of barley Hordeum vulgare and wheat Triticum aestivum.
  • SEQ ID NO: 113 Nucleotide sequence of the protein coding region of a cDNA corresponding to the barley gene encoding ABA-OH-2 (Accession No. DQ145933).
  • SEQ ID NO: 114 Nucleotide sequence of a chimeric DNA encoding a ledRNA construct targeting the ABA-OH-2 genes of barley Hordeum vulgare and wheat Triticum aestivum.
  • SEQ ID NO: 115 Nucleotide sequence of the protein coding region of a cDNA corresponding to the A. thaliana gene encoding EIN2 (At5g03280).
  • SEQ ID NO: 116 Nucleotide sequence of a chimeric DNA encoding a ledRNA construct targeting the EIN2 gene of A. thaliana.
  • SEQ ID NO: 117 Nucleotide sequence of the protein coding region of a cDNA corresponding to the A. thaliana gene encoding CHS (Accession No. NM_121396).
  • SEQ ID NO: 118 Nucleotide sequence of a chimeric DNA encoding a ledRNA construct targeting the CHS gene of A. thaliana.
  • SEQ ID NO: 119 Nucleotide sequence of the protein coding region of a cDNA corresponding to the L. angustifolius N-like gene (Accession No. XM_019604347).
  • SEQ ID NO: 120 Nucleotide sequence of a chimeric DNA encoding a ledRNA construct targeting the L. angustifolius N-like gene.
  • SEQ ID NO: 121 Nucleotide sequence of the protein coding region of a cDNA corresponding to a Vitis pseudoreticulata MLO gene (Accession No. KR362912).
  • SEQ ID NO: 122 Nucleotide sequence of a chimeric DNA encoding a first ledRNA construct targeting a Vitis MLO gene.
  • SEQ ID NO: 123 Nucleotide sequence of the protein coding region of the cDNA corresponding to the MpC002 gene of Myzus persicae.
  • SEQ ID NO: 124 Nucleotide sequence of the protein coding region of the cDNA corresponding to the MpRack-1 gene of Myzus persicae.
  • SEQ ID NO: 125 Nucleotide sequence of the chimeric construct encoding the ledRNA targeting M. persicae C002 gene.
  • SEQ ID NO: 126 Nucleotide sequence of the chimeric construct encoding the ledRNA targeting M. persicae Rack- 1 gene.
  • SEQ ID NO: 127 Nucleotide sequence of the cDNA corresponding to the Helicoverpa armigera ABCwhite gene.
  • SEQ ID NO: 128 Nucleotide sequence of a chimeric DNA encoding a ledRNA construct targeting a ABC transporter white gene of Helicoverpa armigera.
  • SEQ ID NO: 129 Nucleotide sequence of the cDNA corresponding to the Linepithema humile PBAN-type neuropeptides-like (XM_012368710).
  • SEQ ID NO: 130 Nucleotide sequence of a chimeric DNA encoding a ledRNA construct targeting a PBAN gene in Argentine ants (Accession No. XM_012368710).
  • SEQ ID NO: 131 Nucleotide sequence of a chimeric DNA encoding a ledRNA construct targeting a gene encoding V-type proton ATPase catalytic subunit A (Accession No. XM_023443547) of L. cuprina.
  • SEQ ID NO: 132 Nucleotide sequence of a chimeric DNA encoding a ledRNA construct targeting a gene encoding RNAse 1/2 of L. cuprina.
  • SEQ ID NO: 133 Nucleotide sequence of a chimeric DNA encoding a ledRNA construct targeting a gene encoding chitin synthase of L. cuprina.
  • SEQ ID NO: 134 Nucleotide sequence of a chimeric DNA encoding a ledRNA construct targeting a gene encoding ecdysone receptor (EcR) of L. cuprina.
  • SEQ ID NO: 135 Nucleotide sequence of a chimeric DNA encoding a ledRNA construct targeting a gene encoding gamma-tubulin 1/1 -like of L. cuprina.
  • SEQ ID NO: 136 TaMlo target gene (AF384144).
  • SEQ ID NO: 137 Nucleotide sequence of a chimeric DNA encoding a ledRNA construct targeting a gene encoding TaMlo.
  • SEQ ID NO: 138 Nucleotide sequence of the protein coding region of a cDNA corresponding to a Vitis pseudoreticulata MLO gene (Accession No. KR362912).
  • SEQ ID NO: 139 Nucleotide sequence of a chimeric DNA encoding a first ledRNA construct targeting a Vitis MLO gene.
  • SEQ ID NO:140 - Cyp51 homolog 1 (Accession No. KK764651.1, locus RSAG8_00934).
  • SEQ ID NO:141 - Cyp51 homolog 2 (Accession No. KK764892.1, locus number RSAG8_12664).
  • SEQ ID NO: 142 Nucleotide sequence of a chimeric DNA encoding a ledRNA construct targeting a gene encoding Cyp51.
  • SEQ ID NO:143 - CesA3 target gene (Accession No. JN561774.1).
  • SEQ ID NO: 144 Nucleotide sequence of a chimeric DNA encoding a ledRNA construct targeting a gene encoding CesA3.
  • SEQ ID NO: 150 Sequence encoding the LedVRN2 molecule.
  • SEQ ID NO: 151 Nucleotide sequence of the cDNA for Triticum aestivum cultivar Chinese Spring VRN-A1 cDNA protein coding sequence (TaVRNl-Al, Accession No. KR422423.1).
  • SEQ ID NO: 152 Nucleotide sequence of the cDNA for Triticum aestivum flowering locus T cDNA sequence (TaFT, Accession No. AY705794.1).
  • the protein coding sequence is nucleotides 19-549.
  • SEQ ID NO: 153 Nucleotide sequence of the cDNA sequence for Hordeum vulgare subsp. spontaneum MADS box transcription factor (HvVRNl, Accession No. AY896051) gene.
  • the protein coding sequence is nucleotides 8-403.
  • SEQ ID NO: 154 Nucleotide sequence of the cDNA for Hordeum vulgare cultivar Dairokkaku ZCCT-Hb (HvVRN2, Accession No. AY485978) gene, partial cDNA.
  • SEQ ID NO: 156 Nucleotide sequence of the cDNA for Oryza sativa Japonica Group phytochrome B-like gene, transcript variant XI (OsPhyB, LOC4332623, OSNPB_030309200).
  • SEQ ID NO: 157 Nucleotide sequence of the cDNA for Oryza sativa Constans-like 4 gene, OsCol4 protein (Accession No. HC084637).
  • SEQ ID NO: 158 Nucleotide sequence of the cDNA sequence of the Oryza sativa Japonica Group protein RFT1 homolog (OsRFTl, LOC4343254, OSNPB_070486100) gene.
  • the protein coding sequence is nucleotides 167-1753.
  • SEQ ID NO: 159 Nucleotide sequence of the cDNA sequence of the Oryza sativa AP2-like ethylene-responsive transcription factor TOE3 OsSNB (OSNPB_070235800).
  • the protein coding sequence is nucleotides 213-1520.
  • SEQ ID NO: 160 Nucleotide sequence of the cDNA sequence for Oryza sativa Japonica Group AP2-like ethylene-responsive transcription factor TOE3 gene, transcript variant XI, (OsIDSl, LOC4334582, Os03g0818800).
  • the protein coding sequence is nucleotides 575-1876.
  • SEQ ID NO: 161 Nucleotide sequence of the cDNA sequence for Oryza sativa Japonica Group GIGANTEA-like gene, transcript variant XI, (OsGI, LOC4325329, OSNPB_010182600).
  • the protein coding sequence is nucleotides 440-3919.
  • SEQ ID NO: 162 Nucleotide sequence of the cDNA sequence for Oryza sativa OsMADS50 (homolog of AtSOCl) (HC084627).
  • the protein coding sequence is nucleotides 23-712.
  • SEQ ID NO: 163 Nucleotide sequence of the cDNA sequence for Oryza sativa Japonica Group OsMADS55 (homolog of AtSOCl) (Accession No. AY345223).
  • SEQ ID NO: 164 Nucleotide sequence of the cDNA sequence for Oryza sativa Japonica Group transcription factor FL (OsLFY, LOC4336857, 0s04g0598300).
  • the protein coding sequence is nucleotides 233-1399.
  • SEQ ID NO: 165 Nucleotide sequence of the cDNA sequence of gene encoding Zea mays cultivar Assiniboine ZmMADSl/ZmM5 (LOC542042, Accession No. HM993639), partial sequence.
  • SEQ ID NO: 166 Nucleotide sequence of the cDNA sequence of gene encoding Zea mays cultivar B73 phytochrome A1 apoprotein PHYA1 (Accession No. AY234826). Protein coding region is nucleotides 118-3510.
  • SEQ ID NO: 167 Nucleotide sequence of the cDNA sequence of gene encoding Zea mays phytochrome A2 apoprotein PHYA2 (LOCI 15101004, Accession No.
  • Protein coding region is nucleotides 141-3533.
  • SEQ ID NO: 168 Nucleotide sequence of the cDNA sequence of gene encoding Zea mays phytochrome B1 apoprotein PHYB1 (LOC100383702, Accession No.
  • Protein coding region is nucleotides 1-3483.
  • SEQ ID NO: 169 - Nucleotide sequence of the cDNA sequence of gene encoding Zea mays phytochrome B2 apoprotein PHYB2 (Accession No. AY234828). Protein coding region is nucleotides 1-3498.
  • SEQ ID NO: 170 Nucleotide sequence of the cDNA sequence of gene encoding Zea mays phytochrome Cl apoprotein PHYC1 (Accession No. AY234829). Protein coding region is nucleotides 48-3455.
  • SEQ ID NO: 171 Nucleotide sequence of the cDNA sequence of gene encoding Zea mays phytochrome C2 apoprotein PHYC2 (Accession No. AY234830). Protein coding region is nucleotides 141-3533.
  • SEQ ID NO: 172 Nucleotide sequence of the cDNA sequence of gene encoding Zea mays flowering-time protein isoforms alpha and beta (ZmLD), alternatively spliced products (Accession No. AF166527). Protein coding region is nucleotides 122-3669. SEQ ID NO: 173 - Nucleotide sequence of the cDNA sequence of gene encoding Zea mays cultivar A632 floricaula/leafy-like 1 (ZmFLl) (Accession No. AY179882). Protein coding region is nucleotides 27-1199.
  • SEQ ID NO: 174 Nucleotide sequence of the cDNA of gene encoding Zea mays cultivar A632 floricaula/leafy-like 2 (ZmFL2) (Accession No. AY789023).
  • SEQ ID NO: 175 Nucleotide sequence of the cDNA sequence of gene encoding Zea mays cultivar A554 DWARF8 gene (Accession No. AF413203), partial cDNA.
  • SEQ ID NO: 176 Nucleotide sequence of the cDNA sequence of gene encoding Zea mays kaurene synthase A (ZmANl protein, Accession No. L37750). Protein coding region is nucleotides 105-2573.
  • SEQ ID NO: 177 Nucleotide sequence of the cDNA sequence of gene encoding Zea mays zinc finger protein ID1 (ZmIDl protein, Accession No. AF058757). Protein coding region is nucleotides 112-1419.
  • SEQ ID NO: 178 Nucleotide sequence of the cDNA sequence of gene encoding Zea mays ZCN8 (ZmCN8 protein, LOCI 00127519). Protein coding region is nucleotides 60-672.
  • SEQ ID NO: 179 Nucleotide sequence of the cDNA for Brassica napus MADS-box (FLC1) protein gene (BnFLCl-AlO, Accession No. AY036888, BnaA10g22080D).
  • the protein coding sequence is nucleotides 68-658.
  • SEQ ID NO: 180 Nucleotide sequence of the cDNA for Brassica napus MADS-box protein (FLC2) gene (BnFLC2, Accession No. AY036889).
  • the protein coding sequence is nucleotides 34-621.
  • SEQ ID NO: 181 Nucleotide sequence of the cDNA for Brassica napus MADS-box protein (FLC3) (BnFLC3, Accession No. AY036890).
  • the protein coding sequence is nucleotides 46-636.
  • SEQ ID NO: 182 Nucleotide sequence of the cDNA for Brassica napus MADS-box protein (FFC4) (BnFFC4, Accession No. AY036891).
  • the protein coding sequence is nucleotides 147-734.
  • SEQ ID NO: 183 Nucleotide sequence of the cDNA for Brassica napus MADS-box protein (FLC5) (BnFLC5, Accession No. AY036892).
  • the protein coding sequence is nucleotides 63-736.
  • SEQ ID NO: 184 Nucleotide sequence of the cDNA for Brassica napus Frigida gene (BnFRI, BnaA03gl3320D).
  • SEQ ID NO: 185 Nucleotide sequence of the cDNA for Brassica napus linkage group A2 flowering locus T (FT) gene (BnFT, BnaA02gl2130D).
  • SEQ ID NO: 186 Nucleotide sequence of the cDNA sequence for Medicago truncatula cultivar Jester FTal protein (MtFTal, Accession No. HQ721813) gene.
  • the protein coding sequence is nucleotides 233-1399.
  • SEQ ID NO: 187 Nucleotide sequence of the cDNA sequence for Medicago truncatula cultivar Jester FTbl protein (MtFTbl, Accession No. HQ721815) gene.
  • the protein coding sequence is nucleotides 233-1399.
  • SEQ ID NO: 188 Nucleotide sequence of the cDNA sequence for Medicago sativa Frigida-like protein mRNA, (MsFRI-L, Accession No. JX173068, Chao et ah, 2013).
  • the protein coding sequence is nucleotides 7-1563.
  • SEQ ID NO: 189 Nucleotide sequence of the cDNA sequence for Medicago sativa subsp. caerulea shatterproof mRNA, (MsSOCla/ McaeSHP; Accession No. JX297565).
  • the protein coding sequence is from nucleotide 31.
  • SEQ ID NO: 190 Nucleotide sequence of the cDNA sequence for Medicago sativa FT (FT) gene, (MsFT, Accession No. JF681135).
  • SEQ ID NO: 191 Nucleotide sequence of the cDNA sequence for Glycine max MADS-box protein FLOWERING LOCUS C (GmFLC) encoded by the gene GLYMA_05G148700 (Accession No. XMJ314775674, LOC 100804540), transcript variant XI, mRNA.
  • the protein coding sequence is nucleotides 90-686.
  • SEQ ID NO: 192 Nucleotide sequence of the cDNA sequence for Glycine max MADS-box protein FLOWERING LOCUS C encoded by the gene GLYMA_05G148700 (Accession No. XM_003524857.4), transcript variant X2.
  • the protein coding sequence is nucleotides 72-665.
  • SEQ ID NO: 193 Nucleotide sequence of the cDNA sequence for Glycine max MADS -box protein FLOWERING LOCUS C encoded by the gene
  • GLYMA_05G148700 (Accession No. XR_001388453), transcript variant X3.
  • the protein coding sequence is nucleotides 90-653.
  • GLYMA_05G148700 (Accession No. XM_006580064), transcript variant X4.
  • the protein coding sequence is nucleotides 90-641.
  • GLYMA_05G148700 (Accession No. XM_006580065), transcript variant X5.
  • the protein coding sequence is nucleotides 90-605.
  • GLYMA_05G148700 (Accession No. XR_414429.3), transcript variant X6.
  • the protein coding sequence is nucleotides 90-587.
  • GLYMA_05G148700 (Accession No. XM_014775675), transcript variant X7.
  • the protein coding sequence is nucleotides 90-587.
  • GLYMA_05G148700 (Accession No. XM_014775676), transcript variant X8.
  • the protein coding sequence is nucleotides 90-587.
  • GLYMA_05G148700 (Accession No. XM_006580067), transcript variant X9.
  • the protein coding sequence is nucleotides 90-575.
  • SEQ ID NO:200 Nucleotide sequence of the cDNA sequence of gene encoding Glycine max protein SUPPRESSOR OF FRI 4 (LOCI 00819009), transcript variant X3. (Accession No. XM_003530888).
  • the protein coding sequence is nucleotides 145- 1257.
  • SEQ ID NO:201 Nucleotide sequence of the cDNA sequence of gene encoding Glycine max protein FRIGID A-like protein 4a (GmFRI4a, LOC 100805780, Accession No. NM_001360372).
  • the protein coding sequence is nucleotides 77-1828.
  • SEQ ID NO:202 Nucleotide sequence of the cDNA sequence of gene encoding Glycine max protein protein FLOWERING LOCUS T (FT2A, GLYMA_16G150700, Accession No. NM_001253256).
  • the protein coding sequence is nucleotides 78-605.
  • SEQ ID NO:203 Nucleotide sequence of the cDNA sequence of gene encoding Glycine max protein phytochrome A, transcript variant X3 (GmPhyA3, Accession No. XM_014771785.2).
  • the protein coding sequence is nucleotides 615-3899.
  • SEQ ID NO:204 Nucleotide sequence of the cDNA sequence of gene encoding Glycine max protein protein GIGANTEA, transcript variant 1 (GmGIGANTEA Accession No. NM_001354790).
  • the protein coding sequence is nucleotides 419-3946.
  • SEQ ID NO:205 Nucleotide sequence of the cDNA sequence of gene encoding Beta vulgaris subsp. vulgaris genotype KWS2320 bolting time control 1 (BTC1, Accession No. HQ709091). Protein coding region is nucleotides 307-2670.
  • SEQ ID NO:206 Nucleotide sequence of the cDNA sequence of gene encoding Beta vulgaris flowering locus T-like protein (FT1) gene (BvFTl, Accession No.
  • SEQ ID NO:208 Nucleotide sequence of the cDNA sequence of gene encoding Brassica rapa cultivar IMB 218dh FLC2 (FLC2, Accession No. AH012704), partial sequence.
  • SEQ ID NO:209 Nucleotide sequence of the cDNA sequence of gene encoding Brassica rapa FRIGIDA (FRI, Accession No. HQ615935).
  • SEQ ID NO:210 Nucleotide sequence of the cDNA sequence of Medicago truncatula clone MT YFL_FM_FN_FO 1 G-C- 11 (MtYFL, Accession No. BT053010). Protein coding region is nucleotides 78-1136.
  • SEQ ID NO:211 Nucleotide sequence of the cDNA sequence of Allium cepa GIGANTEA (Gla) (AcGIa, Accession No. GQ232756). Protein coding region is nucleotides 27-3353.
  • SEQ ID NO:212 Nucleotide sequence of the cDNA sequence of Allium cepa FKF1 (FKF1, Accession No. GQ232754). Protein coding region is nucleotides 53-1905.
  • SEQ ID NO:213 Nucleotide sequence of the cDNA sequence of Allium cepa ZEITLUPE (AcZTL, Accession No. GQ232755). Protein coding region is nucleotides 128-1963.
  • SEQ ID NO:214 Nucleotide sequence of the cDNA sequence of Allium cepa ACABR20 CONSTANS-like protein (AcCOL, Accession No. GQ232751). Protein coding region is nucleotides 22-972.
  • SEQ ID NO:215 Nucleotide sequence of the cDNA sequence of Allium cepa ACAEE96 protein (AcFTL, Accession No. CF438000). Protein coding region is nucleotides 396-818.
  • SEQ ID NO:216 Nucleotide sequence of the cDNA sequence of Allium cepa cultivar CUDH2150 FT1 (AcFTl, Accession No. KC485348). Protein coding region is nucleotides 1-534.
  • SEQ ID NO:217 Nucleotide sequence of the cDNA sequence of Allium cepa cultivar CUDH2150 FT2 (AcFT2, Accession No. KC485349). Protein coding region is nucleotides 42-566.
  • SEQ ID NO:218 Nucleotide sequence of the cDNA sequence of Allium cepa cultivar CUDH2150 FT6 (AcFT6, Accession No. KC485353). Protein coding region is nucleotides 6-560.
  • SEQ ID NO:219 Nucleotide sequence of the cDNA sequence of Allium cepa clone ACAGK28 phytochrome A (PHYA) (AcPHYA, Accession No. GQ232753), partial sequence. Protein coding region is nucleotides 1-1119.
  • SEQ ID NO:220 Nucleotide sequence of the cDNA sequence of Allium cepa clone ACADQ29 COP1 (AcCOPl, Accession No. CF451443). Protein coding region is nucleotides 249-647.
  • SEQ ID NO:221 Nucleotide sequence of the cDNA sequence of Lactuca sativa protein HEADING DATE 3A-like protein (LsFT, LOCI 11907824). Protein coding region is nucleotides 71-595.
  • SEQ ID NO:222 Nucleotide sequence of the cDNA sequence of Lactuca sativa protein MOTHER of FT and TFLl-like (LsFLl-like, LOCI 11903066, Accession No. XM_023898861).
  • SEQ ID NO:223 Nucleotide sequence of the cDNA sequence of Lactuca sativa protein MOTHER of FT and TFL1 homolog 1-like (LsTFLl, LOCI 11903054, Accession No. XM_023898849).
  • SEQ ID NO:224 Nucleotide sequence of the cDNA sequence of Lactuca sativa FLC (LsFLC, LOCI 11876490, Accession No. JI588382).
  • SEQ ID NO:225 Nucleotide sequence of the cDNA sequence of Lactuca sativa MADS-box protein SOCl-like (LsSOCl, LOCI 11912847, Accession No. XM_023908569). Protein coding region is nucleotides 159-809. SEQ ID NO:226 - Nucleotide sequence of the cDNA sequence of Lactuca sativa MADS-box protein SOCl-like (LsSOCl-like, LOCI 11880753, Accession No. XM_023877169), transcript variant XI. Protein coding region is nucleotides 129-782.
  • SEQ ID NO:227 Nucleotide sequence of the cDNA sequence of Lactuca sativa MADS-box protein SOCl-like (LsSOCl-like, LOCI 11878575). Protein coding region is nucleotides 166-819.
  • SEQ ID NO:228 Nucleotide sequence of the cDNA sequence of Lactuca sativa floricaula/leafy homolog (LsLFY, LOCI 11892192, Accession No. XM_023888266). Protein coding region is nucleotides 1-1278.
  • antisense regulatory element or “antisense ribonucleic acid sequence” or “antisense RNA sequence” as used herein means an RNA sequence that is at least partially complementary to at least a part of a target RNA molecule to which it hybridizes.
  • an antisense RNA sequence modulates (increases or decreases) the expression or amount of a target RNA molecule or its activity, for example through reducing translation of the target RNA molecule.
  • an antisense RNA sequence alters splicing of a target pre-mRNA resulting in a different splice variant.
  • antisense sequences include, but are not limited to, oligonucleotides, oligonucleosides, oligonucleotide analogues, oligonucleotide mimetics, and chimeric combinations of these.
  • antisense activity is used in the context of the present disclosure to refer to any detectable and/or measurable activity attributable to the hybridization of an antisense RNA sequence to its target RNA molecule. Such detection and/or measuring may be direct or indirect. In an embodiment, antisense activity is assessed by detecting and or measuring the amount of target RNA molecule transcript. Antisense activity may also be detected as a change in a phenotype associated with the target RNA molecule.
  • target RNA molecule refers to a gene transcript that is modulated by an antisense RNA sequence according to the present disclosure. Accordingly, “target RNA molecule” can be any RNA molecule the expression or activity of which is capable of being modulated by an antisense RNA sequence. Exemplary target RNA molecules include, but are not limited to, RNA (including, but not limited to pre-mRNA and mRNA or portions thereof) transcribed from DNA encoding a target protein, rRNA, tRNA, small nuclear RNA, and miRNA, including their precursor forms. The target RNA may be the genomic RNA of a plant, or an RNA molecule derived therefrom.
  • the target RNA molecule can be an RNA from an endogenous gene (or mRNA transcribed from the gene) or a gene which is introduced or may be introduced into the plant cell whose expression is associated with a particular phenotype, trait, disorder or disease state, or a nucleic acid molecule from an infectious agent.
  • the target RNA molecule is in a plant cell.
  • the target RNA molecule encodes a protein.
  • antisense activity can be assessed by detecting and or measuring the amount of target protein, for example through its activity such as enzyme activity, or a function other than as an enzyme, or through a phenotype associated with its function.
  • the term “target protein” refers to a protein that is modulated by an antisense RNA sequence according to the present disclosure.
  • antisense activity is assessed by detecting and/or measuring the amount of target RNA molecules and/or cleaved target RNA molecules and/or alternatively spliced target RNA molecules.
  • Antisense activity can be detected or measured using various methods. For example, antisense activity can be detected or assessed by comparing activity in a particular sample and comparing the activity to that of a control sample.
  • targeting is used in the context of the present disclosure to refer to the association of an antisense RNA sequence to a particular target RNA molecule or a particular region of nucleotides within a target RNA molecule.
  • an antisense RNA sequence according to the present disclosure shares complementarity with at least a region of a target RNA molecule.
  • complementarity refers to a sequence of ribonucleotides that is capable of base pairing with a sequence of ribonucleotides on a target RNA molecule, through hydrogen bonding between bases on the ribonucleotides.
  • adenine (A) is complementary to uracil (U) and guanine (G) to cytosine (C).
  • “complementary base” refers to a ribonucleotide of an antisense RNA sequence that is capable of base pairing with a ribonucleotide of a sense RNA sequence in an RNA molecule of the invention or of its target RNA molecule.
  • a ribonucleotide at a certain position of an antisense RNA sequence is capable of hydrogen bonding with a ribonucleotide at a certain position of a target RNA molecule, then the position of hydrogen bonding between the antisense RNA sequence and the target RNA molecule is considered to be complementary at that ribonucleotide.
  • non-complementary refers to a pair of ribonucleotides that do not form hydrogen bonds with one another or otherwise support hybridization.
  • complementary can also be used to refer to the capacity of an antisense RNA sequence to hybridize to another nucleic acid through complementarity.
  • an RNA sequence and its target are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by ribonucleotides that can bond with each other to allow stable association between the antisense RNA sequence and a sense RNA sequence in the RNA molecule of the invention and/or the target RNA molecule.
  • antisense RNA sequence that may comprise up to about 20% nucleotides that are mismatched (i.e., are not complementary to the corresponding nucleotides of the target).
  • the antisense compounds Preferably contain no more than about 15%, more preferably not more than about 10%, most preferably not more than 5% or no mismatches.
  • the remaining ribonucleotides are complementary or otherwise do not disrupt hybridization (e.g., G:U or A:G pairs) between the antisense RNA sequence and the sense RNA sequence or the target RNA molecule.
  • RNA sequence s described herein are at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% (fully) complementary to at least a region of a target RNA molecule.
  • RNA molecules of the invention is used herein to refer to RNA molecules and chimeric RNA molecules.
  • an RNA molecule of the invention can be a chimeric RNA molecule.
  • chimeric RNA molecule refers to any RNA molecule that is not naturally found in nature.
  • chimeric RNA molecules disclosed herein have been modified to create mismatches in region(s) of dsRNA.
  • chimeric RNA molecules may be modified to convert cytosines to uracils.
  • chimeric RNA molecules have been modified via treatment with bisulfite for a time and under conditions sufficient to convert non-methylated cytosines to uracils.
  • ribonucleotide combinations can base pair. Both canonical and non-canonical base pairings are contemplated by the present disclosure.
  • a base pairing can comprise A:T or G:C in a DNA molecule or U:A or G:C in an RNA molecule.
  • a base pairing may comprise A:G or G:T or U:G.
  • canonical base pairing means base pairing between two nucleotides which are A:T or G:C for deoxyribonucleotides or A:U or G:C for ribonucleotides.
  • non-canonical base pairing means an interaction between the bases of two nucleotides other than canonical base pairings, in the context of two DNA or two RNA sequences.
  • non-canonical base pairing includes pairing between G and U (G:U) or between A and G (A:G).
  • Examples of non-canonical base pairing include purine - purine or pyrimidine - pyrimidine. Most commonly in the context of this disclosure, the non-canonical base pairing is G:U.
  • Other examples of non-canonical base pairs, less preferred, are A:C, G:T, G:G and A:A.
  • RNA components that “hybridize” across a series of ribonucleotides refers to RNA components that “hybridize” across a series of ribonucleotides.
  • Those of skill in the art will appreciate that terms such as “hybridize” and “hybridizing” are used to describe molecules that anneal based on complementary nucleic acid sequences. Such molecules need not be 100% complementary in order to hybridize (i.e. they need not “fully base pair”). For example, there may be one or more mismatches in sequence complementarity.
  • stringent hybridization conditions refers to parameters with which the art is familiar, including the variation of the hybridization temperature with length of an RNA molecule.
  • Ribonucleotide hybridization parameters may be found in references which compile such methods, Sambrook, et al. (supra), and Ausubel, et al. (supra).
  • stringent hybridization conditions can refer to hybridization at 65°C in hybridization buffer (3.5xSSC, 0.02% Ficoll, 0.02% polyvinyl pyrrolidone, 0.02% Bovine Serum Albumin (BSA), 2.5 mM NaFhPC (pH7), 0.5% SDS, 2 mM EDTA), followed by one or more washes in 0.2.xSSC, 0.01% BSA at 50°C.
  • low stringency hybridization conditions refers to parameters with which the art is familiar, including the variation of the hybridization temperature with length of an RNA molecule.
  • low stringency hybridization conditions can refer to hybridization at 42°C in hybridization buffer (3.5xSSC, 0.02% Ficoll, 0.02% polyvinyl pyrrolidone, 0.02% Bovine Serum Albumin (BSA), 2.5 mM NaH 2 P0 4 (pH7), 0.5% SDS, 2 mM EDTA), followed by one or more washes in 0.2.xSSC, 0.01% BSA at 30°C.
  • BSA Bovine Serum Albumin
  • the present invention also encompasses RNA components that “fully base pair” across contiguous ribonucleotides.
  • the term “fully base pair” is used in the context of the present disclosure to refer to a series of contiguous ribonucleotide base pairings. A fully base paired series of contiguous ribonucleotides does not comprise gaps or non- basepaired nucleotides within the series.
  • the term “contiguous” is used to refer to a series of ribonucleotides. Ribonucleotides comprising a contiguous series will be joined by a continuous series of phosphodiester bonds, each ribonucleotide being directly bonded to the next.
  • RNA molecules of the present invention comprise a sense sequence and a corresponding antisense sequence.
  • the relationship between these sequences is defined herein.
  • the sequence relationship and activity of the antisense sequence in relation to a target RNA molecule is also defined herein.
  • covalently linked is used in the context of the present disclosure to refer to the link between the first and second RNA components or any RNA sequences or ribonucleotides.
  • a covalent link or bond is a chemical bond that involves the sharing of electron pairs between atoms.
  • the first and second RNA components or the sense RNA sequence and the antisense RNA sequence are covalently linked as part of a single RNA strand which may fold back on itself through self-complementarity.
  • the components are covalently linked across one or more ribonucleotides by phosphodiester bonds.
  • hybridization means the pairing of complementary polynucleotides through basepairing of complementary bases. While not limited to a particular mechanism, the most common mechanism of pairing involves hydrogen bonding, which may be Watson-Crick hydrogen bonding, between complementary ribonucleotides.
  • the phrase “the RNA molecule reduces the target gene activity in the plant cell” or similar phrases means that the target gene transcript is present in the plant cell and exposure or contact of the cell expressing the target gene transcript to the target RNA molecule results in reduced levels and/or activity of the target gene transcript when compared to the same cell lacking the RNA molecule.
  • the target RNA molecule encodes a protein important for flowering.
  • the RNA molecule can have a modulating effect on flowering by the plant.
  • the modulating effect can be early flowering.
  • the modulating effect can be late flowering.
  • RNA molecules according to the present disclosure and compositions comprising the same can be administered to a plant.
  • the term “unrelated in sequence to a target” refers to molecules having less than 50% identity along the full-length of the intervening RNA sequence.
  • the term “related in sequence to a target” refers to molecules having 50% or more identity along the full-length of the intervening RNA sequence.
  • non-transgenic refers to plants that have not been modified by genetic engineering methods.
  • a "control” or “control plant” or “control plant cell” provides a reference point for measuring changes in phenotype of a subject plant or plant cell to which a RNA molecule disclosed herein has been delivered.
  • the control plant or plant cell is a genetically similar plant or plant cell lacking an RNA molecule disclosed herein, preferably an isogenic plant or plant cell.
  • a control may be a single plant or a group of plants or a crop. Identification of a suitable control to provide a reference point for measuring changes in phenotype is considered well within the purview of those of skill in the art.
  • RNA molecules herein that “modulate the timing of plant flowering” are RNA molecules that are able to increase or decrease the time to flowering of a plant.
  • RNA molecules disclosed herein direct early flowering in plants compared to a control(s).
  • RNA molecules disclosed herein direct late flowering in plants compared to a control(s).
  • Flowering time of plants can be assessed by counting the number of days ("time to flower") between sowing or transplanting and the emergence of a first inflorescence.
  • the "flowering time" of a plant can be determined using the method as described in WO 2007/093444.
  • flowering time can be measured indirectly based on the number of rosette leaves before bolting.
  • time of flower and related terms has the common meaning in the art for each plant type being considered and is typically determined by visual inspection of the plant. The particular feature that indicates the onset of flowering may be different for different plant species. It generally means that the first flower of the plant opens or is fertilisable if the flower does not open. For grasses such as wheat, barley and rice, for example, the term “flowering” means that heads or (panicles) emerge.
  • earsly flowering or “early flowering time” are used herein to refer to plants which start to flower earlier than control plants. Hence these terms refer to plants that show an earlier start of flowering.
  • terms such as “late flowering” or “late flowering time” are used herein to refer to plants which start to flower later than control plants. Hence these terms refer to plants that show a later start to flowering.
  • “early flowering” and “late flowering” can be determined by at least a statistically significantly change (decrease or increase) in flowering time compared to a control plant(s) as determined by a two-tailed Student's t-test or other appropriate statistical analysis, P-value ⁇ 0.05.
  • early flowering can refer to a reduction in time to flower by at least about 2 days, 3 days, 5 days, 10 days, 15 days, 20 days, 30 days, 40 days or more.
  • early flowering refers to a reduction in time to flower by at least 5 to 40 days.
  • early flowering refers to a reduction in time to flower by at least 5 to 40 days.
  • early flowering refers to a reduction in time to flower by at least 10 to 30 days.
  • a reduction in time to flower of at least about 2 days, 3 days, 5 days, 10 days, 15 days, 20 days, 30 days or more can indicate early flowering in wheat.
  • a reduction in time to flower of between 5 and 40 days indicates early flowering in wheat.
  • a reduction in time to flower of between 10 and 30 days indicates early flowering in wheat.
  • an early flowering plant has fewer rosette leaves before bolting than control plants.
  • "late flowering" can refer to an increase in time to flower by at least about 2 days, 3 days, 5 days, 10 days, 15 days, 20 days, 30 days, 40 days or more.
  • an increase in time to flower of at least about 2 days, 3 days, 5 days, 10 days, 15 days, 20 days, 30 days, 40 days or more can indicate late flowering in wheat.
  • a late flowering plant has fewer rosette leaves before bolting than control plants.
  • vernalization refers to a process by which flowering is accelerated in plants via exposure of the plant or seed from which the plant is grown to a temperature stimulus or an artificial equivalent.
  • the artificial equivalent is delivering RNA molecule(s) described herein to a plant or a plant part, for example to seed.
  • a “target RNA or gene that modulates the timing of plant flowering” or an “RNA molecule that modulates the timing of plant flowering” is a target RNA, gene or RNA molecule which is involved in the genetic control of flowering in a plant and/or which influences, regulates or modulates the timing of flowering, including affecting the age or developmental stage of a plant at which it flowers and including genes which are involved in sensing environmental cues that lead to promotion or suppression of flowering.
  • long-day conditions refers to photoperiodic conditions where a dark period in a day is shorter than a threshold dark period required for photoperiodic responses (critical dark period). A 14-hour light/ 10-hour dark photoperiod is typically used as a long-day condition.
  • Plants included in the invention are any flowering plants, including both monocotyledonous and dicotyledonous plants.
  • monocotyledonous plants include, but are not limited to, cereals such as wheat, barley, maize, rice, sorghum, pearl millet, rye and oats, grasses such as forage grasses and turfgrasses, vegetables such as asparagus, onions and garlic.
  • dicotyledonous plants include, but are not limited to, vegetables such as such as tomato, legumes such as alfalfa, beans, peas, chickpeas, lupins and soybeans, peppers, lettuce, forage or feed plants such as alfalfa, clover, Brassica species e.
  • the term about refers to +/- 20%, more preferably +/- 10%, of the designated value.
  • RNA molecules of the present invention comprise a first RNA component which is covalently linked to a second RNA component.
  • the RNA molecule self-hybridizes or folds to form a “dumbbell” or ledRNA structure, for example see Figure 1.
  • the molecule further comprises one or more of the following: a linking ribonucleotide sequence which covalently links the first and second RNA components; a 5’ leader sequence; and, a 3’ trailer sequence.
  • the first RNA component consists of, in 5’ to 3’ order, a first 5’ ribonucleotide, a first RNA sequence and a first 3’ ribonucleotide, wherein the first 5’ and 3’ ribonucleotides basepair to each other in the RNA molecule, wherein the first RNA sequence comprises a first sense ribonucleotide sequence of at least 20 contiguous ribonucleotides, a first loop sequence of at least 4 ribonucleotides and a first antisense ribonucleotide sequence of at least 20 contiguous ribonucleotides, wherein the first antisense ribonucleotide sequence hybridises with the first sense ribonucleotide sequence in the RNA molecule, wherein the first antisense ribonucleotide sequence is capable of hybridising to a first region of a target RNA molecule which modulates the timing of plant flowering.
  • the first RNA component consists of, in 5’ to 3’ order, a first 5’ ribonucleotide, a first RNA sequence and a first 3’ ribonucleotide, wherein the first 5’ and 3’ ribonucleotides basepair to each other in the RNA molecule, wherein the first RNA sequence comprises a first sense ribonucleotide sequence of at least 20 contiguous ribonucleotides, a first loop sequence of at least 4 ribonucleotides and a first antisense ribonucleotide sequence of at least 20 contiguous ribonucleotides, wherein the first antisense ribonucleotide sequence fully basepairs with the first sense ribonucleotide sequence in the RNA molecule, wherein the first antisense ribonucleotide sequence is identical in sequence to the complement of a first region of a target RNA molecule.
  • the first RNA component consists of a first 5’ ribonucleotide, a first RNA sequence and a first 3’ ribonucleotide, wherein the first 5’ and 3’ ribonucleotides basepair with each other in the first RNA component, wherein the first RNA sequence comprises a first sense ribonucleotide sequence, a first loop sequence of at least 4 ribonucleotides and a first antisense ribonucleotide sequence, wherein the first sense ribonucleotide sequence and first antisense ribonucleotide sequence each of at least 20 contiguous ribonucleotides whereby the at least 20 contiguous ribonucleotides of the first sense ribonucleotide sequence fully basepair with the at least 20 contiguous ribonucleotides of the first antisense ribonucleotide sequence, wherein the at least 20 contiguous ribon
  • the basepair formed between the first 5’ ribonucleotide and the first 3’ ribonucleotide is considered to be the terminal basepair of the dsRNA region formed by self-hybridization of the first RNA component, i.e it defines the end of the dsRNA region.
  • the first sense sequence has substantial sequence identity to a region of the target RNA, which identity may be to a sequence of less than 20 nucleotides in length. In an embodiment at least 15, at least 16, at least 17, at least 18, or at least 19 contiguous ribonucleotides, preferably at least 20 contiguous ribonucleotides, of the first sense ribonucleotide sequence and a first region of a target RNA molecule are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% identical in sequence.
  • the at least 15, at least 16, at least 17, at least 18, at least 19 contiguous ribonucleotides of the first sense ribonucleotide sequence and a first region of a target RNA molecule are 100% identical.
  • the first 3, first 4, first 5, first 6, or first 7 ribonucleotides from the 5’ end of the first sense ribonucleotide sequence are 100% identical to the region of the target RNA molecule, with the remaining ribonucleotides being at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the target RNA molecule.
  • the at least 20 contiguous ribonucleotides of the first sense ribonucleotide sequence and a first region of a target RNA molecule are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
  • the first 3, first 4, first 5, first 6, or first 7 ribonucleotides can be 100% identical to the region of the target RNA molecule, with the remaining ribonucleotides being at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to target RNA molecule.
  • the at least 20 contiguous ribonucleotides of the first sense ribonucleotide sequence and a first region of a target RNA molecule are 100% identical.
  • the first antisense sequence has substantial sequence identity to the complement of a region of the target RNA, which identity may be to a sequence of less than 20 nucleotides in length of the complement.
  • at least 15, at least 16, at least 17, at least 18, or at least 19 contiguous ribonucleotides, preferably at least 20 contiguous ribonucleotides, of the first antisense ribonucleotide sequence and the complement of a first region of a target RNA molecule are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% identical in sequence.
  • the at least 15, at least 16, at least 17, at least 18, at least 19 contiguous ribonucleotides of the first antisense ribonucleotide sequence and the complement of the first region of the target RNA molecule are 100% identical.
  • the first 3, first 4, first 5, first 6, or first 7 ribonucleotides from the 5’ end of the first antisense ribonucleotide sequence are 100% identical to the complement of the region of the target RNA molecule, with the remaining ribonucleotides being at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the complement of the target RNA molecule.
  • the at least 20 contiguous ribonucleotides of the first antisense ribonucleotide sequence and the complement of a first region of the target RNA molecule are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
  • the first 3, first 4, first 5, first 6, or first 7 ribonucleotides are 100% identical to the complement of the region of the target RNA molecule, with the remaining ribonucleotides being at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the complement of the target RNA molecule.
  • the at least 20 contiguous ribonucleotides of the first antisense ribonucleotide sequence and a first region of a target RNA molecule are 100% identical.
  • the second RNA component consists of, in 5’ to 3’ order, a second 5’ ribonucleotide, a second RNA sequence and a second 3’ ribonucleotide, wherein the second 5’ and 3’ ribonucleotides basepair, wherein the second RNA sequence comprises a second sense ribonucleotide sequence, a second loop sequence of at least 4 ribonucleotides and a second antisense ribonucleotide sequence, wherein the second sense ribonucleotide sequence basepairs with the second antisense ribonucleotide sequence.
  • the basepair formed between the second 5’ ribonucleotide and the second 3’ ribonucleotide is considered to be the terminal basepair of the dsRNA region formed by self-hybridization of the second RNA component.
  • the RNA molecule comprises a 5’ leader sequence, or 5’ extension sequence, which may arise as a result of transcription from a promoter in the genetic construct, from the start site of transcription to the beginning of the polynucleotide encoding the remainder of the RNA molecule. It is preferred that this 5’ leader sequence or 5’ extension sequence is relatively short compared to the remainder of the molecule, and it may be removed from the RNA molecule post-transcriptionally, for embodiment by RNAse treatment.
  • the 5’ leader sequence or 5’ extension sequence may be mostly non-basepaired, or it may contain one or more stem-loop structures.
  • the 5’ leader sequence can consist of a sequence of ribonucleotides which is covalently linked to the first 5’ ribonucleotide if the second RNA component is linked to the first 3’ ribonucleotide or to the second 5’ ribonucleotide if the second RNA component is linked to the first 5’ ribonucleotide.
  • the 5’ leader sequence is at least 10, at least 20, at least 30, at least 100, at least 200 ribonucleotides long, preferably to a maximum length of 250 ribonucleotides.
  • the 5’ leader sequence is at least 50 ribonucleotides long.
  • the 5’ leader sequence can act as an extension sequence for amplification of the RNA molecule via a suitable amplification reaction.
  • the extension sequence may facilitate amplification via polymerase.
  • the RNA molecule comprises a 3’ trailer sequence or 3’ extension sequence which may arise as a result of transcription continuing until a transcription termination or polyadenylation signal in the construct encoding the RNA molecule.
  • the 3’ trailer sequence or 3’ extension sequence may comprise a polyA tail. It is preferred that this 3’ trailer sequence or 3’ extension sequence is relatively short compared to the remainder of the molecule, and it may be removed from the RNA molecule post-transcriptionally, for embodiment by RNAse treatment.
  • the 3’ trailer sequence or 3’ extension sequence may be mostly non-basepaired, or it may contain one or more stem- loop structures.
  • the 3’ trailer sequence can consist of a sequence of ribonucleotides which is covalently linked to the second 3’ ribonucleotide if the second RNA component is linked to the first 3’ ribonucleotide or to the first 3’ ribonucleotide if the second RNA component is linked to the first 5’ ribonucleotide.
  • the 3’ leader sequence is at least 10, at least 20, at least 30, at least 100, at least 200 ribonucleotides long, preferably to a maximum length of 250 ribonucleotides.
  • the 3’ leader sequence is at least 50 ribonucleotides long.
  • the 3’ trailer sequence can act as an extension sequence for amplification of the RNA molecule via a suitable amplification reaction.
  • the extension sequence may facilitate amplification via polymerase.
  • all except for two of the ribonucleotides are covalently linked to two other nucleotides i.e. the RNA molecule consists of only one RNA strand which has self-complementary regions, and so has only one 5’ terminal nucleotide and one 3’ terminal nucleotide.
  • all except for four of the ribonucleotides are covalently linked to two other nucleotides i.e. the RNA molecule consists of two RNA strands which have complementary regions which hybridise, and so has only two 5’ terminal nucleotides and two 3’ terminal nucleotides.
  • each ribonucleotide is covalently linked to two other nucleotides i.e the RNA molecule is circular as well as having self-complementary regions, and so has no 5’ terminal nucleotide and no 3’ terminal nucleotide.
  • the double- stranded region of the RNA molecule can comprise one or more bulges resulting from unpaired nucleotides in the sense RNA sequence or the antisense RNA sequence, or both.
  • the RNA molecule comprises a series of bulges.
  • the double- stranded region of the RNA molecule may have 2, 3, 4, 5, 6, 7, 8, 9, 10 or more bulges.
  • Each bulge may be, independently, one, two or more unpaired nucleotides, to as many as 10 nucleotides. Longer sequences may loop out of the sense or antisense sequences in the dsRNA region, which may basepair internally or remain unpaired.
  • the double-stranded region of the RNA molecule does not comprise a bulge i.e. is fully basepaired along the full length of the dsRNA region.
  • the first sense ribonucleotide sequence is covalently linked to the first 5’ ribonucleotide without any intervening nucleotides, or the first antisense ribonucleotide sequence is covalently linked to the first 3’ ribonucleotide without any intervening nucleotides, or both.
  • the 20 consecutive nucleotides of the first sense ribonucleotide sequence are covalently linked to the first 5’ ribonucleotide without any intervening nucleotides
  • the 20 consecutive nucleotides of the first antisense ribonucleotide sequence are covalently linked to the first 3’ ribonucleotide without any intervening nucleotides.
  • the intervening nucleotides may be basepaired as part of the double-stranded region of the RNA molecule but are unrelated in sequence to the target RNA. They may assist in providing increased stability to the double-stranded region or to hold together two ends of the RNA molecule and not leave an unbasepaired 5’ or 3’ end, or both.
  • the above referenced first and second RNA components comprise a linking ribonucleotide sequence.
  • the linking ribonucleotide sequence acts as a spacer between the first sense ribonucleotide sequence that is substantially identical in sequence to a first region of a target RNA molecule and the other components of the molecule.
  • the linking ribonucleotide sequence may act as a spacer between this region and a loop.
  • the RNA molecule comprises multiple sense ribonucleotide sequences that are substantially identical in sequence to a first region of a target RNA molecule and a linking ribonucleotide sequence which acts as a spacer between these sequences.
  • At least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 10 ribonucleotide sequences that are substantially identical in sequence to a first region of a target RNA molecule are provided in the RNA molecule, each being separated from the other(s) by a linking ribonucleotide sequence.
  • the above referenced RNA molecules comprise a 5’ leader sequence.
  • the 5’ leader sequence consists of a sequence of ribonucleotides which is covalently linked to the first 5’ ribonucleotide if the second RNA component is linked to the first 3’ ribonucleotide or to the second 5’ ribonucleotide if the second RNA component is linked to the first 5’ ribonucleotide.
  • the RNA molecule has a modified 5’ or 3’ end, for embodiment by attachment of a lipid group such as cholesterol, or a vitamin such as biotin, or a polypeptide. Such modifications may assist in the uptake of the RNA molecule into the plant cell where the RNA is to function.
  • the linking ribonucleotide sequence is less than 100 ribonucleotides in length. In an embodiment, the linking ribonucleotide sequence is less than 50 ribonucleotides in length. In an embodiment, the linking ribonucleotide sequence is less than 20 ribonucleotides in length. In an embodiment, the linking ribonucleotide sequence is less than 10 ribonucleotides in length. In an embodiment, the linking ribonucleotide sequence is less than 5 ribonucleotides in length. In an embodiment, the linking ribonucleotide sequence is between 1 and 100 ribonucleotides in length.
  • the linking ribonucleotide sequence is between 1 and 50 ribonucleotides in length. In an embodiment, the linking ribonucleotide sequence is between 1 and 20 ribonucleotides in length. In an embodiment, the linking ribonucleotide sequence is between 1 and 10 ribonucleotides in length. In an embodiment, the linking ribonucleotide sequence is between 1 and 5 ribonucleotides in length. In an embodiment, the ribonucleotides of the linking ribonucleotide sequence are not basepaired. In a preferred embodiment, the ribonucleotides of the linking ribonucleotide sequence are all basepaired, or all except for 1, 2 or 3 of the ribonucleotides are basepaired.
  • the first or second RNA component comprises a hairpin structure.
  • the first and second RNA components each comprise a hairpin structure.
  • the hairpin structure can be a stem-loop.
  • the RNA molecule can comprise first and second RNA components which each comprise a hairpin structure, wherein the hairpins are covalently bound by a linker sequence. See, for example, Figure 1.
  • the linker sequence is one or more unpaired ribonucleic acid(s). In an embodiment, the linker sequence is between 1 and 10 unpaired ribonucleotides.
  • the RNA molecule has a double hairpin structure i.e. an “ledRNA structure” or “dumbbell structure”.
  • the first hairpin is the first RNA component and the second hairpin is the second RNA component.
  • either the first 3’ ribonucleotide and the second 5’ ribonucleotide, or the second 3’ ribonucleotide and the first 5’ ribonucleotide, but not both, are covalently joined.
  • the other 573’ ribonucleotides can be separated by a nick (i.e.
  • the respective 573’ ribonucleotides can be separated by a loop.
  • the lengths of the 5’ leader and 3’ trailer sequences may be the same or different.
  • the 5’ leader may be around 5, 10, 15, 20, 25, 50, 100, 200, 500 ribonucleotides longer than the 3’ trailer sequence or vice versa.
  • the second hairpin (in addition to the first hairpin structure) comprises a sense RNA sequence and an antisense RNA sequence that are substantially identical in sequence to a region of a target RNA molecule or its complement, respectively.
  • each hairpin has a series of ribonucleotides that are substantially identical in sequence to a region of the same target RNA molecule.
  • each hairpin has a series of ribonucleotides that are substantially identical in sequence to different regions of the same target RNA molecule.
  • each hairpin has a series of ribonucleotides that are substantially identical in sequence to a region of different target RNA molecules i.e. the RNA molecule can be used to reduce the expression and/or activity of two target RNA molecules which may be unrelated in sequence.
  • the order of the sense and antisense RNA sequences in each hairpin may independently be either sense then antisense, or antisense then sense.
  • the order of the sense and antisense sequences in the double hairpin structure of the RNA molecule is either antisense-sense-sense-antisense where the two sense sequences are contiguous ( Figure 1A), or sense-antisense-antisense-sense where the two antisense sequences are contiguous ( Figure IB).
  • the RNA molecule can comprise, in 5’ to 3’ order, a 5’ leader sequence, a first loop, a sense RNA sequence, a second loop and a 3’ trailer sequence, wherein the 5’ and 3’ leader sequences covalently bond to the sense strand to form a dsRNA sequence.
  • the 5’ leader and 3’ trailer sequences are not covalently bound to each other.
  • the 5’ leader and 3’ trailer sequences are separated by a nick.
  • the 5’ leader and 3’ trailer sequences are ligated together to provide a RNA molecule with a closed structure.
  • the 5’ leader and 3’ trailer sequences are separated by a loop.
  • loop is used in the context of the present disclosure to refer to a loop structure in an RNA molecule disclosed herein that is formed by a series of non complementary ribonucleotides. Loops generally follow a series of base-pairs between the first and second RNA components or join a sense RNA sequence and an antisense RNA sequence in one or both of the first and second RNA components. In an embodiment, all of the loop ribonucleotides are non-complementary, generally for shorter loops of 4-10 ribonucleotides. In other embodiments, some ribonucleotides in one or more of the loops are complementary and capable of basepairing within the loop sequence, so long as these basepairings enable a loop structure to form. For example, at least 5%, at least 10%, or at least 15% of the loop ribonucleotides are complementary. Embodiments of loops include stem loops or hairpins, pseudoknots and tetraloops.
  • the RNA molecule comprises only two loops, In another embodiment, the RNA molecule comprises at least two, at least three, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 loops, preferably to a maximum of 10 loops.
  • the RNA molecule can comprise 4 loops.
  • loops of various sizes are contemplated by the present disclosure.
  • loops can comprise 4, 5, 6, 7, 8, 9, 10, 11 or 12 ribonucleotides.
  • loops comprise 15, 20, 25 or 30 nucleotides.
  • one or all of the loop sequences are longer than 20 nucleotides.
  • loops are larger, for example comprising 50, 100, 150, 200 or 300 ribonucleotides.
  • loops comprise 160 ribonucleotides.
  • loops comprise 200, 500, 700 or 1,000 ribonucleotides provided that the loops do not interfere with the hybridisation of the sense and antisense RNA sequences.
  • each of the loops have the same number of ribonucleotides.
  • loops can have between 100 and 1,000 ribonucleotides in length.
  • loops can have between 600 and 1,000 ribonucleotides in length.
  • loops can have between 4 and 1,000 ribonucleotides.
  • loops preferably have between 4 and 50 ribonucleotides.
  • loops comprise differing numbers of ribonucleotides.
  • one or more loops comprise an intron which can be spliced out of the RNA molecule.
  • the intron is from a plant gene.
  • Exemplary introns include intron 3 of the maize alcohol dehydrogenase 1 (Adhl) (GenBank: AF044293), intron 4 of the soya beta-conglycinin alpha subunit (GenBank: AB051865); one of the introns of the pea rbcS-3A gene for the ribulose-1,5- bisphosphate carboxylase (RBC) small subunit (GenBank: X04333).
  • RBC ribulose-1,5- bisphosphate carboxylase
  • a loop may be at the end of at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 consecutive basepairs, which may be canonical basepairs or may include one or more non-canonical basepairs.
  • the RNA molecule comprises two or more sense ribonucleotide sequences, and antisense ribonucleotide sequences fully based paired thereto, which are each identical in sequence to a region of a target RNA molecule.
  • the RNA molecule can comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more sense ribonucleotide sequences, and antisense ribonucleotide sequences fully based paired thereto, which sense ribonucleotide sequences are each independently identical in sequence to a region of a target RNA molecule.
  • any one or more or all of the sequences can be separated by a linking ribonucleotide sequence(s).
  • any one or more or all of the sequences can be separated by a loop.
  • the two or more sense ribonucleotide sequences are identical in sequence to different regions of the same target RNA molecule.
  • the sequences can be identical to at least 2, at least 3, at least 4, at least 5, at least 6 regions of the same target molecule.
  • the two or more sense ribonucleotide sequences are identical in sequence.
  • the two or more sense ribonucleotide sequences are identical in sequence to the same region of the same target RNA molecule.
  • the two or more sense ribonucleotide sequences are identical in sequence to different target RNA molecules.
  • the sequences can be identical to at least 2, at least 3, at least 4, at least 5, at least 6 regions of different target molecules.
  • the two or more sense ribonucleotide sequences have no intervening loop (spacer) sequences.
  • the RNA molecule has a single strand of ribonucleotides having a 5’ end, at least one sense ribonucleotide sequence which is at least 21 nucleotides in length, an antisense ribonucleotide sequence which is fully basepaired with each sense ribonucleotide sequence over at least 21 contiguous nucleotides, at least two loop sequences and a 3’ end.
  • the ribonucleotide at the 5’ end and the ribonucleotide at the 3’ end are not directly covalently bonded but are rather positioned adjacent with each basepaired.
  • consecutive basepairs of RNA components are interspaced by at least one gap.
  • the “gap” is provided by an unpaired ribonucleotide.
  • the “gap” is provided by un-ligated 5’ leader sequence and/or 3’ trailer sequence.
  • the gap can be referred to as an “unligated gap”. Mismatches and unligated gap(s) can be located at various position(s) of the RNA molecule.
  • an unligated gap can immediately follow an antisense sequence.
  • an unligated gap can be close to a loop of the RNA molecule.
  • an unligated gap is positioned about equidistant between at least two loops.
  • the RNA molecule is produced from a single strand of RNA.
  • the single strand is not circularly closed, for example, comprising an unligated gap.
  • the RNA molecule is a circularly closed molecule. Closed molecules can be produced by ligating an above referenced RNA molecule comprising an unligated gap, for example with an RNA ligase.
  • the RNA molecule comprises a 5’- or 3’-, or both, extension sequence.
  • the RNA molecule can comprise a 5’ extension sequence which is covalently linked to the first 5’ ribonucleotide.
  • the RNA molecule comprises a 3’ extension sequence which is covalently linked to the second 3’ ribonucleotide.
  • the RNA molecule comprises a 5’ extension sequence which is covalently linked to the first 5’ ribonucleotide and a 3’ extension sequence which is covalently linked to the second 3’ ribonucleotide.
  • the RNA molecule comprises a 5’ extension sequence which is covalently linked to the second 5’ ribonucleotide. In another embodiment, the RNA molecule comprises a 3’ extension sequence which is covalently linked to the first 3’ ribonucleotide. In another embodiment, the RNA molecule comprises a 5’ extension sequence which is covalently linked to the second 5’ ribonucleotide and a 3’ extension sequence which is covalently linked to the first 3’ ribonucleotide.
  • RNA molecule can comprise one or more of the following:
  • 3’ extension sequence which is covalently linked to the first 3’ ribonucleotide; a 5’ extension sequence which is covalently linked to the second 5’ ribonucleotide and a 3’ extension sequence which is covalently linked to the first 3’ ribonucleotide.
  • the RNA molecule comprises a nucleic acid sequence set forth in SEQ ID NO: 146 or SEQ ID NO: 147.
  • RNA molecules of the present invention comprise a sense ribonucleotide sequence and an antisense ribonucleotide sequence which are capable of hybridising to each other to form a double stranded (ds)RNA region with some non- canonical basepairing i.e. with a combination of canonical and non-canonical basepairing.
  • RNA molecules of the present invention comprise two or more sense ribonucleotide sequences which are each capable of hybridising to regions of one (contiguous) antisense ribonucleotide sequence to form a dsRNA region with some non-canonical basepairing. See for example, Figure IB.
  • RNA molecules of the present invention comprise two or more antisense sense ribonucleotide sequences which are each capable of hybridising to regions of one (contiguous) sense ribonucleotide sequence to form a dsRNA region with some non- canonical basepairing. See for example, Figure 1A.
  • RNA molecules of the present invention comprise two or more antisense sense ribonucleotide sequences and two or more sense ribonucleotide sequences wherein each antisense ribonucleotide sequence is capable of hybridising to an antisense ribonucleotide sequence to form two or more dsRNA regions, one or both comprising some non- canonical basepairing.
  • the full length of the dsRNA region (i.e. the whole dsRNA region) of the RNA molecule of the invention is considered as the context for the feature if there is only one (contiguous) dsRNA region, or for each of the dsRNA regions of the RNA molecule if there are two or more dsRNA regions in the RNA molecule.
  • at least 5% of the basepairs in a dsRNA region are non-canonical basepairs.
  • at least 6% of the basepairs in a dsRNA region are non-canonical basepairs.
  • At least 7% of the basepairs in a dsRNA region are non-canonical basepairs. In an embodiment, at least 8% of the basepairs in a dsRNA region are non-canonical basepairs. In an embodiment, at least 9% or 10% of the basepairs in a dsRNA region are non-canonical basepairs. In an embodiment, at least 11% or 12% of the basepairs in a dsRNA region are non- canonical basepairs. In an embodiment, at least 15% or about 15% of the basepairs in a dsRNA region are non-canonical basepairs.
  • At least 20% or about 20% of the basepairs in a dsRNA region are non-canonical basepairs. In an embodiment, at least 25% or about 25% of the basepairs in a dsRNA region are non- canonical basepairs. In an embodiment, at least 30% or about 30% of the basepairs in a dsRNA region are non-canonical basepairs.
  • a maximum of 40% of the basepairs in the dsRNA region are non- canonical basepairs, more preferably a maximum of 35% of the basepairs in the dsRNA region are non-canonical basepairs, still more preferably a maximum of 30% of the basepairs in the dsRNA region are non-canonical basepairs. In an embodiment, less preferred, about 35% of the basepairs in a dsRNA region are non-canonical basepairs. In an embodiment, even less preferred, about 40% of the basepairs in a dsRNA region are non-canonical basepairs.
  • the dsRNA region may or may not comprise one or more non-basepaired ribonucleotides, in either the sense sequence or the antisense sequence, or both.
  • between 10% and 40% of the basepairs in a dsRNA region of the RNA molecule of the invention are non-canonical basepairs. In an embodiment, between 10% and 35% of the basepairs in a dsRNA region are non-canonical basepairs. In an embodiment, between 10% and 30% of the basepairs in a dsRNA region are non- canonical basepairs. In an embodiment, between 10% and 25% of the basepairs in a dsRNA region are non-canonical basepairs. In an embodiment, between 10% and 20% of the basepairs in a dsRNA region are non-canonical basepairs.
  • between 10% and 15% of the basepairs in a dsRNA region are non-canonical basepairs. In an embodiment, between 15% and 30% of the basepairs in a dsRNA region are non- canonical basepairs. In an embodiment, between 15% and 25% of the basepairs in a dsRNA region are non-canonical basepairs. In an embodiment, between 15% and 20% of the basepairs in a dsRNA region are non-canonical basepairs. In an embodiment, between 5% and 30% of the basepairs in a dsRNA region are non-canonical basepairs.
  • the dsRNA region may or may not comprise one or more non-basepaired ribonucleotides, in either the sense sequence or the antisense sequence, or both.
  • the dsRNA region of the RNA molecule of the invention comprises 20 contiguous basepairs, wherein at least one basepair of the 20 contiguous basepairs is a non-canonical basepair. In an embodiment, the dsRNA region comprises
  • the dsRNA region comprises 20 contiguous basepairs, wherein at least 2 basepairs of the 20 contiguous basepairs are non-canonical basepairs.
  • the dsRNA region comprises 20 contiguous basepairs, wherein at least 3 basepairs of the 20 contiguous basepairs are non-canonical basepairs.
  • the dsRNA region comprises 20 contiguous basepairs, wherein at least 4 basepairs of the 20 contiguous basepairs are non-canonical basepairs.
  • the dsRNA region comprises 20 contiguous basepairs, wherein at least 5 basepairs of the 20 contiguous basepairs are non-canonical basepairs.
  • the dsRNA region comprises 20 contiguous basepairs, wherein at least 6 basepairs of the 20 contiguous basepairs are non-canonical basepairs. In an embodiment, the dsRNA region comprises 20 contiguous basepairs, wherein at least 7 basepairs of the 20 contiguous basepairs are non-canonical basepairs. In an embodiment, the dsRNA region comprises 20 contiguous basepairs, wherein at least 8 basepairs of the 20 contiguous basepairs are non-canonical basepairs.
  • the dsRNA region comprises 20 contiguous basepairs, wherein at least 9 basepairs of the 20 contiguous basepairs are non-canonical basepairs.
  • a maximum of 10 of the 20 contiguous basepairs in the dsRNA region are non-canonical basepairs, more preferably a maximum of 9 of the basepairs in the dsRNA region are non- canonical basepairs, still more preferably a maximum of 8 of the basepairs in the dsRNA region are non-canonical basepairs, even still more preferably a maximum of 7 of the basepairs in the dsRNA region are non-canonical basepairs, and most preferably a maximum of 6 of the basepairs in the dsRNA region are non-canonical basepairs.
  • the non-canonical basepairs comprise at least one G:U basepair, more preferably all of the non-canonical basepairs are G:U basepairs.
  • the features of the above embodiments apply to each and every one of the 20 contiguous basepairs that are present in the RNA molecule of the invention.
  • the dsRNA region of the RNA molecule of the invention comprises 21 contiguous basepairs, wherein at least one basepair of the 21 contiguous basepairs is a non-canonical basepair. In an embodiment, the dsRNA region comprises
  • the dsRNA region comprises 21 contiguous basepairs, wherein at least 2 basepairs of the 21 contiguous basepairs are non-canonical basepairs.
  • the dsRNA region comprises 21 contiguous basepairs, wherein at least 3 basepairs of the 21 contiguous basepairs are non-canonical basepairs.
  • the dsRNA region comprises 21 contiguous basepairs, wherein at least 4 basepairs of the 21 contiguous basepairs are non-canonical basepairs.
  • the dsRNA region comprises 21 contiguous basepairs, wherein at least 5 basepairs of the 21 contiguous basepairs are non-canonical basepairs.
  • the dsRNA region comprises 21 contiguous basepairs, wherein at least 6 basepairs of the 21 contiguous basepairs are non-canonical basepairs. In an embodiment, the dsRNA region comprises 21 contiguous basepairs, wherein at least 7 basepairs of the 21 contiguous basepairs are non-canonical basepairs. In an embodiment, the dsRNA region comprises 21 contiguous basepairs, wherein at least 8 basepairs of the 21 contiguous basepairs are non-canonical basepairs.
  • the dsRNA region comprises 21 contiguous basepairs, wherein at least 9 basepairs of the 21 contiguous basepairs are non-canonical basepairs.
  • a maximum of 10 of the 21 contiguous basepairs in the dsRNA region are non-canonical basepairs, more preferably a maximum of 9 of the basepairs in the dsRNA region are non- canonical basepairs, still more preferably a maximum of 8 of the basepairs in the dsRNA region are non-canonical basepairs, even still more preferably a maximum of 7 of the basepairs in the dsRNA region are non-canonical basepairs, and most preferably a maximum of 6 of the basepairs in the dsRNA region are non-canonical basepairs.
  • the non-canonical basepairs comprise at least one G:U basepair, more preferably all of the non-canonical basepairs are G:U basepairs.
  • the features of the above embodiments apply to each and every one of the 21 contiguous basepairs that are present in the RNA molecule of the invention.
  • the dsRNA region of the RNA molecule of the invention comprises 22 contiguous basepairs, wherein at least one basepair of the 22 contiguous basepairs is a non-canonical basepair. In an embodiment, the dsRNA region comprises 22 contiguous basepairs, wherein at least 2 basepairs of the 22 contiguous basepairs are non-canonical basepairs. In an embodiment, the dsRNA region comprises 22 contiguous basepairs, wherein at least 3 basepairs of the 22 contiguous basepairs are non-canonical basepairs.
  • the dsRNA region comprises 22 contiguous basepairs, wherein at least 4 basepairs of the 22 contiguous basepairs are non-canonical basepairs. In an embodiment, the dsRNA region comprises 22 contiguous basepairs, wherein at least 5 basepairs of the 22 contiguous basepairs are non-canonical basepairs. In an embodiment, the dsRNA region comprises 22 contiguous basepairs, wherein at least 6 basepairs of the 22 contiguous basepairs are non-canonical basepairs.
  • the dsRNA region comprises 22 contiguous basepairs, wherein at least 7 basepairs of the 22 contiguous basepairs are non-canonical basepairs. In an embodiment, the dsRNA region comprises 22 contiguous basepairs, wherein at least 8 basepairs of the 22 contiguous basepairs are non-canonical basepairs. In an embodiment, the dsRNA region comprises 22 contiguous basepairs, wherein at least 9 basepairs of the 22 contiguous basepairs are non-canonical basepairs.
  • a maximum of 10 of the 22 contiguous basepairs in the dsRNA region are non-canonical basepairs, more preferably a maximum of 9 of the basepairs in the dsRNA region are non- canonical basepairs, still more preferably a maximum of 8 of the basepairs in the dsRNA region are non-canonical basepairs, even still more preferably a maximum of 7 of the basepairs in the dsRNA region are non-canonical basepairs, and most preferably a maximum of 6 of the basepairs in the dsRNA region are non-canonical basepairs.
  • the non-canonical basepairs comprise at least one G:U basepair, more preferably all of the non-canonical basepairs are G:U basepairs.
  • the features of the above embodiments apply to each and every one of the 22 contiguous basepairs that are present in the RNA molecule of the invention.
  • the dsRNA region of the RNA molecule of the invention comprises 23 contiguous basepairs, wherein at least one basepair of the 23 contiguous basepairs is a non-canonical basepair. In an embodiment, the dsRNA region comprises 23 contiguous basepairs, wherein at least 2 basepairs of the 23 contiguous basepairs are non-canonical basepairs. In an embodiment, the dsRNA region comprises 23 contiguous basepairs, wherein at least 3 basepairs of the 23 contiguous basepairs are non-canonical basepairs.
  • the dsRNA region comprises 23 contiguous basepairs, wherein at least 4 basepairs of the 23 contiguous basepairs are non-canonical basepairs. In an embodiment, the dsRNA region comprises 23 contiguous basepairs, wherein at least 5 basepairs of the 23 contiguous basepairs are non-canonical basepairs. In an embodiment, the dsRNA region comprises 23 contiguous basepairs, wherein at least 6 basepairs of the 23 contiguous basepairs are non-canonical basepairs.
  • the dsRNA region comprises 23 contiguous basepairs, wherein at least 7 basepairs of the 23 contiguous basepairs are non-canonical basepairs. In an embodiment, the dsRNA region comprises 23 contiguous basepairs, wherein at least 8 basepairs of the 23 contiguous basepairs are non-canonical basepairs. In an embodiment, the dsRNA region comprises 23 contiguous basepairs, wherein at least 9 basepairs of the 23 contiguous basepairs are non-canonical basepairs.
  • a maximum of 10 of the 23 contiguous basepairs in the dsRNA region are non-canonical basepairs, more preferably a maximum of 9 of the basepairs in the dsRNA region are non- canonical basepairs, still more preferably a maximum of 8 of the basepairs in the dsRNA region are non-canonical basepairs, even still more preferably a maximum of 7 of the basepairs in the dsRNA region are non-canonical basepairs, and most preferably a maximum of 6 of the basepairs in the dsRNA region are non-canonical basepairs.
  • the non-canonical basepairs comprise at least one G:U basepair, more preferably all of the non-canonical basepairs are G:U basepairs.
  • the features of the above embodiments apply to each and every one of the 23 contiguous basepairs that are present in the RNA molecule of the invention.
  • the dsRNA region of the RNA molecule of the invention comprises 24 contiguous basepairs, wherein at least one basepair of the 24 contiguous basepairs is a non-canonical basepair. In an embodiment, the dsRNA region comprises 24 contiguous basepairs, wherein at least 2 basepairs of the 24 contiguous basepairs are non-canonical basepairs. In an embodiment, the dsRNA region comprises 24 contiguous basepairs, wherein at least 3 basepairs of the 24 contiguous basepairs are non-canonical basepairs.
  • the dsRNA region comprises 24 contiguous basepairs, wherein at least 4 basepairs of the 24 contiguous basepairs are non-canonical basepairs. In an embodiment, the dsRNA region comprises 24 contiguous basepairs, wherein at least 5 basepairs of the 24 contiguous basepairs are non-canonical basepairs. In an embodiment, the dsRNA region comprises 24 contiguous basepairs, wherein at least 6 basepairs of the 24 contiguous basepairs are non-canonical basepairs.
  • the dsRNA region comprises 24 contiguous basepairs, wherein at least 7 basepairs of the 24 contiguous basepairs are non-canonical basepairs. In an embodiment, the dsRNA region comprises 24 contiguous basepairs, wherein at least 8 basepairs of the 24 contiguous basepairs are non-canonical basepairs. In an embodiment, the dsRNA region comprises 24 contiguous basepairs, wherein at least 9 basepairs of the 24 contiguous basepairs are non-canonical basepairs.
  • a maximum of 10 of the 24 contiguous basepairs in the dsRNA region are non-canonical basepairs, more preferably a maximum of 9 of the basepairs in the dsRNA region are non- canonical basepairs, still more preferably a maximum of 8 of the basepairs in the dsRNA region are non-canonical basepairs, even still more preferably a maximum of 7 of the basepairs in the dsRNA region are non-canonical basepairs, and most preferably a maximum of 6 of the basepairs in the dsRNA region are non-canonical basepairs.
  • the non-canonical basepairs comprise at least one G:U basepair, more preferably all of the non-canonical basepairs are G:U basepairs.
  • the features of the above embodiments apply to each and every one of the 24 contiguous basepairs that are present in the RNA molecule of the invention.
  • the full length of the dsRNA region (i.e. the whole dsRNA region) of the RNA molecule of the invention is considered as the context for the feature if there is only one (contiguous) dsRNA region, or for each of the dsRNA regions of the RNA molecule if there are two or more dsRNA regions in the RNA molecule.
  • the dsRNA region does not comprise 20 contiguous canonical basepairs i.e. every subregion of 20 contiguous basepairs includes at least one non-canonical basepair, preferably at least one G:U basepair.
  • the dsRNA region does not comprise 19 contiguous canonical basepairs.
  • the dsRNA region does not comprise 18 contiguous canonical basepairs. In an embodiment, the dsRNA region does not comprise 17 contiguous canonical basepairs. In an embodiment, the dsRNA region does not comprise 16 contiguous canonical basepairs. In an embodiment, the dsRNA region does not comprise 15 contiguous canonical basepairs. In an embodiment, the dsRNA region does not comprise 14 contiguous canonical basepairs. In an embodiment, the dsRNA region does not comprise 13 contiguous canonical basepairs. In an embodiment, the dsRNA region does not comprise 12 contiguous canonical basepairs.
  • the dsRNA region does not comprise 11 contiguous canonical basepairs. In an embodiment, the dsRNA region does not comprise 10 contiguous canonical basepairs. In an embodiment, the dsRNA region does not comprise 9 contiguous canonical basepairs. In an embodiment, the dsRNA region does not comprise 8 contiguous canonical basepairs. In an embodiment, the dsRNA region does not comprise 7 contiguous canonical basepairs.
  • the longest subregion of contiguous canonical basepairing in the dsRNA region of the RNA molecule, or each and every dsRNA region in the RNA molecule is 5, 6 or 7 contiguous canonical basepairs i.e. towards the shorter lengths mentioned.
  • the dsRNA region comprises between 10 and 19 or 20 contiguous basepairs.
  • the dsRNA region comprises between 12 and 19 or 20 contiguous basepairs.
  • the dsRNA region comprises between 14 and 19 or 20 contiguous basepairs.
  • the dsRNA region comprises 15 contiguous basepairs. In an embodiment, the dsRNA region comprises 16, 17, 18 or 19 contiguous basepairs. In an embodiment, the dsRNA region comprises 20 contiguous basepairs.
  • the contiguous basepairs comprise at least one non-canonical basepair which comprises at least one G:U basepair, more preferably all of the non- canonical basepairs in the region of contiguous basepairs are G:U basepairs.
  • the dsRNA region comprises a subregion of 4 canonical basepairs flanked by non-canonical basepairs, i.e. at least one, preferably one or two (not more than 2), non-canonical basepairs adjacent to each end of the 4 canonical basepairs.
  • the dsRNA region comprises 2 subregions each of 4 canonical basepairs flanked by non-canonical basepairs.
  • the dsRNA region comprises 3 subregions each of 4 canonical basepairs flanked by non-canonical basepairs.
  • the dsRNA region comprises 4 or 5 subregions each of 4 canonical basepairs flanked by non-canonical basepairs. In an embodiment, the dsRNA region comprises 6 or 7 subregions each of 4 canonical basepairs flanked by non- canonical basepairs. In an embodiment, the dsRNA region comprises 8 to 10 subregions each of 4 canonical basepairs flanked by non-canonical basepairs. In an embodiment, the dsRNA region comprises 11 to 15 subregions each of 4 canonical basepairs flanked by non-canonical basepairs.
  • the dsRNA region comprises between 2 and 50 subregions each of 4 canonical basepairs flanked by non- canonical basepairs. In an embodiment, the dsRNA region comprises between 2 and 40 subregions each of 4 canonical basepairs flanked by non-canonical basepairs. In an embodiment, the dsRNA region comprises between 2 and 30 subregions each of 4 canonical basepairs flanked by non-canonical basepairs. In an embodiment, the dsRNA region comprises between 2 and 20 subregions each of 4 canonical basepairs flanked by non-canonical basepairs.
  • the non-canonical basepairs comprise at least one G:U basepair, more preferably all of the non-canonical basepairs flanking the contiguous canonical basepairs in the subregions are G:U basepairs.
  • one or both of the flanking non- canonical basepairs are replaced with a non-basepaired ribonucleotide in the sense sequence, the antisense sequence or in both sequences, for some or all of the subregions. It is readily understood that, in the above embodiments, the maximum number of subregions is determined by the length of the dsRNA region in the RNA molecule.
  • the dsRNA region comprises a subregion of 5 canonical basepairs flanked by non-canonical basepairs. In an embodiment, the dsRNA region comprises 2 subregions each of 5 canonical basepairs flanked by non-canonical basepairs. In an embodiment, the dsRNA region comprises 3 subregions each of 5 canonical basepairs flanked by non-canonical basepairs. In an embodiment, the dsRNA region comprises 4 or 5 subregions each of 5 canonical basepairs flanked by non- canonical basepairs.
  • the dsRNA region comprises 6 or 7 subregions each of 5 canonical basepairs flanked by non-canonical basepairs. In an embodiment, the dsRNA region comprises 8 to 10 subregions each of 5 canonical basepairs flanked by non-canonical basepairs. In an embodiment, the dsRNA region comprises 11 to 15 subregions each of 5 canonical basepairs flanked by non-canonical basepairs. In an embodiment, the dsRNA region comprises between 2 and 50 subregions each of 5 canonical basepairs flanked by non-canonical basepairs.
  • the dsRNA region comprises between 2 and 50 subregions each of 5 canonical basepairs flanked by non-canonical basepairs. In an embodiment, the dsRNA region comprises between 2 and 30 subregions each of 5 canonical basepairs flanked by non-canonical basepairs. In an embodiment, the dsRNA region comprises between 2 and 20 subregions each of 5 canonical basepairs flanked by non-canonical basepairs.
  • the non-canonical basepairs comprise at least one G:U basepair, more preferably all of the non-canonical basepairs flanking the contiguous canonical basepairs in the subregions are G:U basepairs.
  • one or both of the flanking non-canonical basepairs are replaced with a non-basepaired ribonucleotide in the sense sequence, the antisense sequence or in both sequences, for some or all of the subregions. It is readily understood that, in the above embodiments, the maximum number of subregions is determined by the length of the dsRNA region in the RNA molecule.
  • the dsRNA region comprises a subregion of 6 canonical basepairs flanked by non-canonical basepairs. In an embodiment, the dsRNA region comprises 2 subregions each of 6 canonical basepairs flanked by non-canonical basepairs. In an embodiment, the dsRNA region comprises 3 subregions each of 6 canonical basepairs flanked by non-canonical basepairs. In an embodiment, the dsRNA region comprises 4 or 5 subregions each of 6 canonical basepairs flanked by non- canonical basepairs.
  • the dsRNA region comprises 6 or 7 subregions each of 6 canonical basepairs flanked by non-canonical basepairs. In an embodiment, the dsRNA region comprises 8 to 10 subregions each of 6 canonical basepairs flanked by non-canonical basepairs. In an embodiment, the dsRNA region comprises 11 to 16 subregions each of 6 canonical basepairs flanked by non-canonical basepairs. In an embodiment, the dsRNA region comprises between 2 and 60 subregions each of 6 canonical basepairs flanked by non-canonical basepairs.
  • the dsRNA region comprises between 2 and 60 subregions each of 6 canonical basepairs flanked by non-canonical basepairs. In an embodiment, the dsRNA region comprises between 2 and 30 subregions each of 6 canonical basepairs flanked by non-canonical basepairs. In an embodiment, the dsRNA region comprises between 2 and 20 subregions each of 6 canonical basepairs flanked by non-canonical basepairs.
  • the non-canonical basepairs comprise at least one G:U basepair, more preferably all of the non-canonical basepairs flanking the contiguous canonical basepairs in the subregions are G:U basepairs.
  • one or both of the flanking non-canonical basepairs are replaced with a non-basepaired ribonucleotide in the sense sequence, the antisense sequence or in both sequences, for some or all of the subregions. It is readily understood that, in the above embodiments, the maximum number of subregions is determined by the length of the dsRNA region in the RNA molecule.
  • the dsRNA region comprises a subregion of 10 contiguous basepairs wherein 2-4 of the basepairs are non-canonical basepairs. In an embodiment, the dsRNA region comprises 2 subregions each of 10 contiguous basepairs wherein 2-4 of the 10 contiguous basepairs are non-canonical basepairs. In an embodiment, the dsRNA region comprises 3 subregions each of 10 contiguous basepairs wherein 2-4 of the 10 contiguous basepairs are non-canonical basepairs.
  • the dsRNA region comprises 4 subregions each of 10 contiguous basepairs wherein 2-4 of the 10 contiguous basepairs are non-canonical basepairs. In an embodiment, the dsRNA region comprises 5 subregions each of 10 contiguous basepairs wherein 2-4 of the 10 contiguous basepairs are non-canonical basepairs. In an embodiment, the dsRNA region comprises 10 subregions each of 10 contiguous basepairs wherein 2-4 of the 10 contiguous basepairs are non-canonical basepairs.
  • the dsRNA region comprises 4 subregions each of 15 contiguous basepairs wherein 2-6 of the 15 contiguous basepairs are non-canonical basepairs. In an embodiment, the dsRNA region comprises between 2 and 50 subregions each of 10 contiguous basepairs wherein 2-4 of the 10 contiguous basepairs are non-canonical basepairs. In an embodiment, the dsRNA region comprises between 2 and 40 subregions each of 10 contiguous basepairs wherein 2-4 of the 10 contiguous basepairs are non-canonical basepairs.
  • the dsRNA region comprises between 2 and 30 subregions each of 10 contiguous basepairs wherein 2-4 of the 10 contiguous basepairs are non-canonical basepairs. In an embodiment, the dsRNA region comprises between 2 and 20 subregions each of 10 contiguous basepairs wherein 2-4 of the 10 contiguous basepairs are non-canonical basepairs. In an embodiment, the non-canonical basepairs in one (contiguous) or more, or all dsRNA regions of the RNA molecule are not adjacent a non-base pair. In another embodiment, the non-canonical basepairs are at least 2 continguous base pairs from a non-base pair.
  • the non- canonical basepairs are at least 3, 4, 5, 6, 7, 8, 9, 10 or more continguous base pairs from a non-base pair.
  • the non-canonical basepairs in one (contiguous) or more, or all dsRNA regions of the RNA molecule are not adjacent a loop sequence.
  • the non-canonical basepairs are at least 2 continguous base pairs from a loop sequence.
  • the non- canonical basepairs are at least 3, 4, 5, 6, 7, 8, 9, 10 or more continguous base pairs from a loop sequence.
  • the non-canonical basepairs comprise at least one G:U basepair, more preferably all of the non-canonical basepairs in the subregions are G:U basepairs.
  • one or more of the 2-4 or 2-6 non-canonical basepairs are replaced with a non- basepaired ribonucleotide in the sense sequence, the antisense sequence or in both sequences, for some or all of the subregions. It is readily understood that, in the above embodiments, the maximum number of subregions is determined by the length of the dsRNA region in the RNA molecule.
  • the ratio of canonical to non-canonical basepairs in the dsRNA region is between 2.5:1 and 3.5:1, for example about 3:1. In an embodiment, the ratio of canonical to non-canonical basepairs in the dsRNA region is between 3.5:1 and 4.5:1, for example about 4:1. In an embodiment, the ratio of canonical to non- canonical basepairs in the dsRNA region is between 4.5:1 and 5.5:1, for example about 5:1. In an embodiment, the ratio of canonical to non-canonical basepairs in the dsRNA region is between 5.5:1 and 6.5:1, for example about 6:1. Different dsRNA regions in the RNA molecule may have different ratios.
  • the non-canonical basepairs in the dsRNA region(s) of the RNA molecule are preferably all G:U basepairs. In an embodiment, at least 99% of the non-canonical basepairs are G:U basepairs. In an embodiment, at least 98% of the non-canonical basepairs are G:U basepairs. In an embodiment, at least 97% of the non-canonical basepairs are G:U basepairs. In an embodiment, at least 95% of the non- canonical basepairs are G:U basepairs. In an embodiment, at least 90% of the non- canonical basepairs are G:U basepairs.
  • between 90 and 95% of the non-canonical basepairs are G:U basepairs. For example, if there are 10 non-canonical basepairs, at least 9 (90%) are G:U basepairs. In another embodiment, between 3% and 50% of the non-canonical basepairs are G:U basepairs. In another embodiment, between 5% and 30% of the non-canonical basepairs are G:U basepairs. In another embodiment, between 10% and 30% of the non-canonical basepairs are G:U basepairs. In another embodiment, between 15% and 20% of the non-canonical basepairs are G:U basepairs.
  • the dsRNA region comprising non-canonical basepairing(s) comprises an antisense sequence of 20 contiguous nucleotides which acts as an antisense regulatory element.
  • the antisense regulatory element is at least 80%, preferably at least 90%, more preferably at least 95% or most preferably 100% complementary to a target RNA molecule in a plant cell.
  • a dsRNA region comprises 2, 3, 4, or 5 antisense regulatory elements which either are complementary to the same target RNA molecule (i.e. to different regions of the same target RNA molecule) or are complementary to different target RNA molecules.
  • one or more ribonucleotides of the sense ribonucleotide sequence or one or more ribonucleotides of the antisense ribonucleotide sequence, or both are not basepaired in the dsRNA region when the sense and antisense sequences hybridize.
  • the dsRNA region does not include any loop sequence which covalently joins the sense and antisense sequences.
  • One or more ribonucleotides of a dsRNA region or subregion may not be basepaired. Accordingly, in this embodiment, the sense strand of the dsRNA region does not fully basepair with its corresponding antisense strand.
  • the chimeric RNA molecule does not comprise a non- canonical base pair at the base of a loop of the molecule. In another embodiment, one, two, three, four, five or more or all of the non-canonical base pairs are flanked by canonical base pairs.
  • the chimeric RNA molecule comprises at least one plant DCL-1 cleavage site.
  • the target RNA molecule is not a viral RNA molecule.
  • the target RNA molecule is not a South African cassava mosaic vims RNA molecule.
  • the chimeric RNA molecule comprises at least one non- basepair, or stretch of non-pasepairs, flanked by canonical base pairs, non-canonical base pairs, or a canonical base pair and a non-canonical base pair. For example, this may be a bulge as described herein.
  • the chimeric RNA molecule does not comprise a double stranded region with greater than 11 canonical base pairs.
  • the total number of ribonucleotides in the sense sequence(s) and the total number of ribonucleotides in the antisense sequence(s) may not be identical, although preferably they are identical.
  • the total number of ribonucleotides in the sense ribonucleotide sequence(s) of the dsRNA region is between 90% and 110% of the total number of ribonucleotides in the antisense ribonucleotide sequence(s).
  • the total number of ribonucleotides in the sense ribonucleotide sequence(s) is between 95% and 105% of the total number of ribonucleotides in the antisense ribonucleotide sequence(s).
  • chimeric RNA molecules of the present disclosure can comprise one or more structural elements such as internal or terminal bulges or loops. Various embodiments of bulges and loops are discussed above.
  • dsRNA regions are separated by a structural element such as a bulge or loop.
  • dsRNA regions are separated by a intervening (spacer) sequence.
  • ribonucleotides of the spacer sequence may be basepaired to other ribonucleotides in the RNA molecule, for example to other ribonucleotides within the spacer sequence, or they may not be basepaired in the RNA molecule, or some of each.
  • dsRNA regions are linked to a terminal loop. In an embodiment, dsRNA regions are flanked by terminal loops.
  • the non-canonical basepairs are not contiguous but are separated by one or more canonical basepairs i.e. the dsRNA region does not have 3 or more contiguous non-canonical basepairs. In an embodiment, the dsRNA region does not have 4 or more contiguous non-canonical basepairs.
  • the dsRNA region comprises at least 3 non-canonical basepairs in a subregion of 10 basepairs, wherein each non-canonical basepair is separated by 4 canonical basepairs.
  • an RNA molecule of the invention comprises more than one dsRNA region.
  • the RNA molecule comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more dsRNA regions.
  • one or more or all of the dsRNA regions can comprise above exemplified properties such as non-canonical basepairing and/or number of antisense regulatory elements.
  • RNA molecules of the present disclosure have antisense activity as they comprise a sense ribonucleotide sequence that is essentially complementary to a region of a target RNA molecule.
  • the ribonucleotide sequence is essentially complementary to a region of a target RNA molecule in a plant cell.
  • Such components of the RNA molecules defined herein can be referred to as an “antisense regulatory element”.
  • “Essentially complementary” means that the sense ribonucleotide sequence may have insertions, deletions and individual point mutations in comparison with the complement of the target RNA molecule in the plant cell.
  • the homology is at least 80%, preferably at least 90%, preferably at least 95%, most preferably 100%, between the sense ribonucleotide sequence with antisense activity and the target RNA molecule.
  • the sense ribonucleotide sequence can comprise about 15, about 16, about 17, about 18, about 19 or more contiguous nucleotides that are identical in sequence to a first region of a target RNA molecule in a plant cell.
  • the sense ribonucleotide sequence can comprise about 20 contiguous nucleotides that are identical in sequence to a first region of a target RNA molecule in a plant cell.
  • Antisense activity is used in the context of the present disclosure to refer to an antisense regulatory element from an RNA molecule defined herein that modulates (increase or decrease) expression of a target RNA molecule.
  • antisense regulatory elements can comprise a plurality of monomeric subunits linked together by linking groups. Examples include primers, probes, antisense compounds, antisense oligonucleotides, external guide sequence (EGS) oligonucleotides, alternate splicers, gapmers, siRNAs and microRNAs.
  • RNA molecules according to the present disclosure can comprise antisense regulatory elements with single-stranded, double- stranded, circular, branched or hairpin structures.
  • the antisense sequence can contain structural elements such as internal or terminal bulges or loops.
  • RNA molecules of the present disclosure comprise chimeric oligomeric components such as chimeric oligonucleotides.
  • an RNA molecule can comprise differently modified nucleotides, mixed-backbone antisense oligonucleotides or a combination thereof.
  • chimeric oligomeric compounds can comprise at least one region modified so as to confer increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target RNA molecule.
  • Antisense regulatory elements can have a variety of lengths. Across various examples, the present disclosure provides antisense regulatory elements consisting of X-Y linked bases, where X and Y are each independently selected from 8, 9, 10, 11, 12,
  • the present disclosure provides antisense regulatory elements comprising: 8-9, 8-10, 8-11, 8-12, 8-13, 8-14, 8-15, 8-16, 8-17, 8-18, 8-19, 8- 20, 8-21, 8-22, 8-23, 8-24, 8-25, 8-26, 8-27, 8-28, 8-29, 8-30, 9-10, 9-11, 9-12, 9-13, 9-
  • RNA molecules according to the present disclosure can comprise multiple antisense regulatory elements.
  • RNA molecules can comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 antisense regulatory elements.
  • the antisense regulatory elements are the same.
  • the RNA molecule can comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 copies of an antisense regulatory element.
  • RNA molecules according to the present disclosure can comprise different antisense regulatory elements.
  • antisense regulatory elements may be provided to target multiple genes in a pathway such as lipid biosynthesis.
  • the RNA molecule can comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 different antisense regulatory elements.
  • Antisense sequences according to the present disclosure can modulate, preferably decrease, expression or amount of various target RNA molecules.
  • the target RNA molecule modulates flowering in a plant disclosed herein. Examples of such target RNA molecules are described in the art (e.g. Cockram et al., 2007; Chen et al., 2009; Jung and Muller., 2009; Cho et al., 2017).
  • the target RNA molecule modulates vernalisation in a plant disclosed herein.
  • the target RNA molecule promotes early flowering.
  • the target RNA molecule promotes late flowering.
  • the target RNA molecule encodes a plant polycomb group (PcG) protein.
  • the target RNA molecule encodes VERNALIZATION1 (VRN1; UniProt accession number: Q8L3W1) or VERNALIZATION2 (VRN2; UniProt accession number: Q8W5B1) or homologous genes in other species.
  • the target RNA molecule encodes a PcG from Arabidopsis, corn, canola, cotton, soybean, alfalfa, lettuce, wheat, barley, rice, legume, Medicago truncatula, sugarbeet or rye.
  • the target RNA molecule encodes a PcG from Arabidopsis, corn, canola, cotton, soybean, wheat, barley, rice, legume, Medicago truncatula, sugarbeet or rye.
  • the target RNA molecule encodes VRN1 and/or VRN2 from wheat.
  • the target RNA molecule encodes EMBRYONIC FLOWER2 (EMF2; UniProt accession number: Q8L6Y4) or FERTILIZATION INDEPENDENT SEED2 (FIS2; UniProt accession number: P0DKJ7) or homologous genes in other species.
  • the target RNA molecule encodes one or more or all of VRN1, VRN2, EMF2, FIS2.
  • Other examples of target RNA molecules encode EARLYINSHORTDAYS4 (ESD4; UniProt accession number: Q94F30) and FLOWERING LOCUS T (FLT; UniProt accession number: Q9SXZ2) or homologous genes in other species.
  • the target RNA molecules can be a gene transcript of one or more of VRN1, VRN2, EMF2, FIS2, ESD4, FFT1, FFT2.
  • the target RNA molecule can be a gene transcript of one or more of the following from wheat/barley, VRN1/VRN-A1 (KR422423.1); VRN2 (ZCCT1, TaVRN-2B) (AAS58481.1); FT (AY705794.1).
  • the target RNA molecule can be a gene transcript of one or more of the following from canola, BnFLCl (AY036888, Bna.FLC.A10, BnaA10g22080D); BnFLC2 (AY036889); BnFLC3 (AY036890); BnFLC4 (AY036891); BnFLC5 (AY036892); BnFRI (BnaA03gl3320D); BnFT (BnaA02gl2130D).
  • the target RNA molecule can be a gene transcript of BnFLCl (AY036888, Bna.FLC.A10, BnaA10g22080D).
  • the target RNA molecule can be a gene transcript of a FRIGIDA orthologue such as BnaA3.FRI (Yi et ah, 2018) or homologous genes in other species.
  • the target RNA molecule can be a gene transcript of one or more of the following from Arabidopsis, FRI (AT4G00650); FLC (AT5G10140); VRN1 (AT3G18990); VRN2 (AT4G16845); VIN3 (AT5G57380); FT (AT1G65480); SOC1 (AT2G45660); CO (constans) (AT5G15840); LFY (AT5G61850); API (AT1G69120) or homologous genes in other species.
  • FRI AT4G00650
  • FLC AT5G10140
  • VRN1 AT3G18990
  • VRN2 AT4G16845
  • VIN3 AT5G57380
  • FT AT1G65480
  • SOC1 AT2G45660
  • the target RNA molecule can be a gene transcript of one or more of the following from Rice, OsPhyB (OSNPB_030309200); OsCol4 (Hd-1) (HC084637); RFT1 (OSNPB_070486100); OsSNB (OSNPB_070235800) ; OsIDSl (0s03g0818800); OsGI (OSNPB_010182600) or homologous genes in other species.
  • the target RNA molecule can be a gene transcript of one or more of the following from Medicago truncatula, MtFTal (HQ721813); MtFTbl (HQ721815) or homologous genes in other species.
  • the target RNA molecule can be a gene transcript of a homolog of one or more of the following from Legume, MtFTal; MtFTbl.
  • the target RNA molecule can be a gene transcript of one or more of the following from Sugarbeet, chard, turnip, BTC1 (T1Q709091.); BvFTl (HM448909.1); BvFLl (DQ189214., DQ189215.) or homologous genes in other species.
  • the target RNA molecule can be a gene transcript of one or more of the following from barley, HvVRNl (AY896051); HvVRN2 (AY687931, AY485978); HvFT (DQ898519) or homologous genes in other species.
  • the target RNA molecule can be a gene transcript of one or more of the following from Maize, ZmMADS l/ZmM5 (LOC542042, HM993639); PHYA1 (AY234826); PHYA2 (AY260865); PHYB1 (AY234827); PHYB2 (AY234828); PHYC1 (AY234829); PHYC2 (AY234830); LD (AF166527); ZFL1 (AY179882); ZFL2 (AY179881); DWARF8 (AF413203); AN1 (L37750); ID1 (AF058757); ZCN8 (LOC100127519) or homologous genes in other species.
  • the target RNA molecule can be a gene transcript of one or more of the following from Brassica rapa, BrFLC2 (AH012704); BrFT (Bra004928); BrFRI (HQ615935) or homologous genes in other species.
  • the target RNA molecule can be a gene transcript of MsFRI-L (JX173068) from Alfalfa (Medicago sativa) or homologous genes in other species.
  • the target RNA molecule can be a gene transcript of one or more of the following from Barrell medic, MtYFL (BT053010); MtSOCla (Medtr07 g075870); MtSOClb (Medtr08g033250); MtSOClc (Medtr08g033220); MtFTal (HQ721813) or homologous genes in other species.
  • MtYFL B053010
  • MtSOCla Medtr07 g075870
  • MtSOClb Medtr08g033250
  • MtSOClc Medtr08g033220
  • MtFTal HQ721813
  • the target RNA molecule can be a gene transcript of one or more of the following from cotton, GhCO (Gorai.008G059900); GhFLC (Gorai.013G069000); GhFRI (Gorai.003Gl 18000); GhFT (Gorai.004G264600); GhLFY (Gorai.001G053900); GhPHYA (Gorai.007G292800, Gorai.013G203900); GhPHYB (Gorai.011G200200); GhSOCl (Gorai.008Gl 15200); GhVRNl (Gorai.002G006500, Gorai.005G240900, Gorai.012G150900, Gorai.013G040000); GhVRN2 (Gorai.003G176300); GhVRN5 (Gorai.009G023200) or homologous genes in other species.
  • GhCO Gorai.008G059900
  • GhFLC Gorai.013G069000
  • the target RNA molecule can be a gene transcript of one or more of the following from onion, AcGI (GQ232756); AcFKF (GQ232754); AcZTL (GQ232755); AcCOL (GQ232751); AcFTL (CF438000); AcFTl (KC485348); AcFT2 (KC485349); AcFT6 (KC485353); AcPHYA (GQ232753); AcCOPl (CF451443) or homologous genes in other species.
  • the target RNA molecule can be a gene transcript of one or more of the following from Asparagus officinalis, FPA (LOC 109824259, LOC109840062); TWIN SISTER of FT-like (LOC109835987); MOTHER of FT (LOC109844838); FCA-like (LOC109841154, LOC109821266); PHOTOPERIOD- INDEPENDENT EARLY FLOWERING 1 (LOC 109834006); FLOWERING LOCUS T-like (LOC 109830558, LOC109825338, LOC109824462); Flowering locus K (LOC109847537); Flowering time control protein FY (LOC109844014); flowering time control protein FCA-like (LOC 109842562) or homologous genes in other species.
  • FPA LOC 109824259, LOC109840062
  • TWIN SISTER of FT-like LOC109835987
  • MOTHER of FT LOC10984
  • the target RNA molecule can be a gene transcript of one or more of the following from lettuce, LsFT (LOCI 11907824); TFLl-like (LOCI 11903066); TFL1 homolog 1-like (LOCI 11903054); LsFLC (LOCI 11876490, JI588382); SOC1- like (LOCI 11912847, LOCI 11880753, LOCI 11878575); TsLFY (LC 164345.1, XM_023888266.1) or homologous genes in other species.
  • LsFT LOCI 11907824
  • TFLl-like LOCI 11903066
  • TFL1 homolog 1-like LOCI 11903054
  • LsFLC LOCI 11876490, JI588382
  • SOC1- like LOCI 11912847, LOCI 11880753, LOCI 11878575
  • TsLFY LC 164345.1, XM_023888266.1
  • the present disclosure extends to homologues thereof. Identifying homologues is considered well within the purview of those skilled in the art using various online databases such as Genbank, EMBL-EBI, Ensembl Plants or performing online searches using tools such as nucleotide BLAST. Examples of homologues are provided above.
  • the target RNA molecule can be a gene transcript of BnFLCl or a homolog thereof such as, for example BnFLCl (AY036888), BnFLCl (Bna.FLC.A10) or BnFLCl (BnaA10g22080D).
  • the target RNA is a non-coding RNA that modulates flowering in plants.
  • the non-coding RNA is a miRNA or pre-cursor thereof.
  • the target miRNA is a miRNA from the miR-156 family or a precursor thereof.
  • the target RNA can be any one or more of miR-156a, miR-156b, miR-156c, miR-156d, miR-156e, miR-156f, miR-156g, miR-156h or a precursor thereof.
  • the target RNA is one or more of miR-156a, miR- 156b, miR-156c or a precursor thereof.
  • the target RNA is miR-172 or a precursor thereof.
  • miRNA sequences are described in the art and can be identified by for example miRBase: the microRNA database (Kozomara et al., 2019); www dot mirbase dot org).
  • the target RNA molecule is a transcript from a VRN2 gene.
  • nucleic acid encoding RNA molecules disclosed herein and the component parts thereof.
  • a nucleic acid comprising a sequence set forth in any one or more of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 150.
  • the nucleic acid may be partially purified after expression in a host cell.
  • partially purified is used to refer to an RNA molecule that has generally been separated from the lipids, nucleic acids, other peptides, and other contaminating molecules with which it is associated in a host cell.
  • the partially purified polynucleotide is at least 60% free, more preferably at least 75% free, and more preferably at least 90% free from other components with which it is associated.
  • a polynucleotide according to the present disclosure is a heterologous polynucleotide.
  • heterologous polynucleotide is well understood in the art and refers to a polynucleotide which is not endogenous to a cell, or is a native polynucleotide in which the native sequence has been altered, or a native polypeptide whose expression is quantitatively altered as a result of a manipulation of the cell by recombinant DNA techniques.
  • a polynucleotide according to the present disclosure is a synthetic polynucleotide.
  • the polynucleotide may be produced using techniques that do not require pre-existing nucleic acid sequences such as DNA printing and oligonucleotide synthesis.
  • the polynucleotide is produced from xeno nucleic acids.
  • a polynucleotide disclosed herein which encodes an RNA precursor molecule comprising an intron, preferably in a 5’ extension sequence or in at least one loop sequence, wherein the intron is capable of being spliced out during transcription of the polynucleotide in a host cell or in vitro.
  • the loop sequence comprises two, three, four, five or more introns.
  • the present disclosure also provides an expression construct such as a DNA construct comprising an isolated nucleic acid of the disclosure operably linked to a promoter.
  • isolated nucleic acids and/or expression constructs are provided in a cell or plant.
  • isolated nucleic acids are stably integrated into the genome of the cell or plant organism.
  • RNA molecules according to the present disclosure can be achieved using various methods known in the art.
  • the Examples section provides an example of in vitro synthesis.
  • constructs comprising RNA molecules disclosed herein are restricted at the 3' end, precipitated, purified and quantified.
  • RNA synthesis can be achieved in bacterial culture following transformation of HT115 electro competent cells and induction of RNA synthesis using the T7, IPTG system.
  • One embodiment of the present invention includes a recombinant vector, which comprises at least one RNA molecule defined herein and is capable of delivering the RNA molecule into a host cell.
  • Recombinant vectors include expression vectors.
  • Recombinant vectors contain heterologous polynucleotide sequences, that is, polynucleotide sequences that are not naturally found adjacent to an RNA molecule defined herein, that preferably, are derived from a different species.
  • the vector can be either RNA or DNA, and typically is a viral vector, derived from a virus, or a plasmid.
  • viral vectors can be used to deliver and mediate expression of an RNA molecule according to the present disclosure.
  • the choice of viral vector will generally depend on various parameters, such as the cell or tissue targeted for delivery, transduction efficiency of the vector and pathogenicity.
  • the viral vector integrates into host cellular chromatin (e.g. lentivimses).
  • the viral vector persists in the cell nucleus predominantly as an extrachromosomal episome (e.g. adenoviruses). Examples of these types of viral vectors include oncoretroviruses, lentivimses, adeno-associated virus, adenoviruses, herpes viruses and retroviruses.
  • Plasmid vectors typically include additional nucleic acid sequences that provide for easy selection, amplification, and transformation of the expression cassette in prokaryotic cells, e.g., pUC-derived vectors, pGEM-derived vectors or binary vectors containing one or more T-DNA regions.
  • Additional nucleic acid sequences include origins of replication to provide for autonomous replication of the vector, selectable marker genes, preferably encoding antibiotic or herbicide resistance, unique multiple cloning sites providing for multiple sites to insert nucleic acid sequences or genes encoded in the nucleic acid construct, and sequences that enhance transformation of plant cells.
  • operably linked refers to a functional relationship between two or more nucleic acid (e.g., DNA) segments. Typically, it refers to the functional relationship of a transcriptional regulatory element (promoter) to a transcribed sequence.
  • a transcriptional regulatory element promoter
  • a promoter is operably linked to a coding sequence of an RNA molecule defined herein, if it stimulates or modulates the transcription of the coding sequence in an appropriate cell.
  • promoter transcriptional regulatory elements that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis- acting.
  • some transcriptional regulatory elements such as enhancers need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.
  • each promoter may independently be the same or different.
  • the recombinant vector desirably comprises a selectable or screenable marker gene.
  • marker gene is meant a gene that imparts a distinct phenotype to cells expressing the marker gene and thus, allows such transformed cells to be distinguished from cells that do not have the marker.
  • a selectable marker gene confers a trait for which one can "select” based on resistance to a selective agent (e.g., a herbicide, antibiotic).
  • a screenable marker gene confers a trait that one can identify through observation or testing, that is, by “screening” (e.g., b-glucuronidase, luciferase, GFP or other enzyme activity not present in untransformed cells).
  • Exemplary selectable markers for selection of plant transformants include, but are not limited to, a hyg gene which encodes hygromycin B resistance; a neomycin phosphotransferase (nptll) gene conferring resistance to kanamycin, paromomycin; a glutathione-S-transferase gene from rat liver conferring resistance to glutathione derived herbicides as for example, described in EP 256223; a glutamine synthetase gene conferring, upon overexpression, resistance to glutamine synthetase inhibitors such as phosphinothricin as for example, described in WO 87/05327; an acetyltransferase gene from Streptomyces viridochromogenes conferring resistance to the selective agent phosphinothricin as for example, described in EP 275957; a gene encoding a 5-enolshikimate-3-phosphate synthase (EPSPS) conferring tolerance to
  • a nitrilase gene such as bxn from Klebsiella ozaenae which confers resistance to bromoxynil (
  • the recombinant vector is stably incorporated into the genome of the cell such as the plant cell.
  • the recombinant vector may comprise appropriate elements which allow the vector to be incorporated into the genome, or into a chromosome of the cell.
  • an "expression vector” is a DNA vector that is capable of transforming a host cell and of effecting expression of an RNA molecule defined herein.
  • Expression vectors of the present invention contain regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the host cell and that control the expression of RNA molecule according to the present disclosure.
  • expression vectors of the present invention include transcription control sequences. Transcription control sequences are sequences which control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation such as promoter, enhancer, operator and repressor sequences. The choice of the regulatory sequences used may depends on the target plant or part therof. Such regulatory sequences may be obtained from any eukaryotic organism such as plants or plant viruses, or may be chemically synthesized.
  • plant expression vectors include for example, one or more cloned plant genes under the transcriptional control of 5' and 3' regulatory sequences and a dominant selectable marker.
  • Such plant expression vectors also can contain a promoter regulatory region (e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally-regulated, or cell- or tissue- specific expression), a transcription initiation start site, a ribosome binding site, a transcription termination site, and/or a polyadenylation signal.
  • a promoter regulatory region e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally-regulated, or cell- or tissue- specific expression
  • Vectors of the invention can also be used to produce RNA molecules defined herein in a cell-free expression system, such systems are well known in the art.
  • a polynucleotide encoding an RNA molecule according to the present disclosure is operably linked to a promoter capable of directing expressing of the RNA molecule in a host cell.
  • the promoter functions in vitro.
  • the promoter is an RNA polymerase promoter.
  • the promoter can be an RNA polymerase III promoter.
  • the promoter can be an RNA polymerase II promoter.
  • the choice of promoter may depend on the target plant or part therof.
  • Exemplary promoters which may be suitable for constitutive expression in plants include, but are not limited to, the cauliflower mosaic virus (CaMV) 35S promoter, the Figwort mosaic virus (FMV) 35S, the light-inducible promoter from the small subunit (SSU) of the ribulose-l,5-bis-phosphate carboxylase, the rice cytosolic triosephosphate isomerase promoter, the adenine phosphoribosyltransferase promoter of Arabidopsis, the rice actin 1 gene promoter, the mannopine synthase and octopine synthase promoters, the Adh promoter, the sucrose synthase promoter, the R gene complex promoter, and the chlorophyll a/b binding protein gene promoter. These promoters have been used to create DNA vectors that have been expressed in plants, see for example, WO 84/02913. All of these promoters have been used to create various types of plant-expressible recomb
  • promoters utilized in the present invention have relatively high expression in these specific tissues.
  • promoters for genes with tissue- or cell- specific, or -enhanced expression. Examples of such promoters reported in the literature include, the chloroplast glutamine synthetase GS2 promoter from pea, the chloroplast fructose- 1,6- biphosphatase promoter from wheat, the nuclear photosynthetic ST-LS1 promoter from potato, the serine/threonine kinase promoter and the glucoamylase (CHS) promoter from Arabidopsis thaliana.
  • CHS glucoamylase
  • ribulose-l,5-bisphosphate carboxylase promoter from eastern larch ( Larix laricina), the promoter for the Cab gene, Cab6, from pine, the promoter for the Cab-1 gene from wheat, the promoter for the Cab-1 gene from spinach, the promoter for the Cab 1R gene from rice, the pyruvate, orthophosphate dikinase (PPDK) promoter from Zea mays , the promoter for the tobacco Lhcbl*2 gene, the Arabidopsis thaliana Suc2 sucrose-H 30 symporter promoter, and the promoter for the thylakoid membrane protein genes from spinach (PsaD, PsaF, PsaE, PC, FNR, AtpC, AtpD, Cab, RbcS).
  • Other promoters for the chlorophyll a/b-binding proteins may also be utilized in the present invention such as the promoters for LhcB
  • RNA-binding protein genes in plant cells, including promoters regulated by heat, light (e.g., pea RbcS-3A promoter, maize RbcS promoter), hormones such as abscisic acid, wounding (e.g., Wunl), or chemicals such as methyl jasmonate, salicylic acid, steroid hormones, alcohol, Safeners (WO 97/06269), or it may also be advantageous to employ organ-specific promoters.
  • heat e.g., pea RbcS-3A promoter, maize RbcS promoter
  • hormones such as abscisic acid
  • wounding e.g., Wunl
  • chemicals e.g., methyl jasmonate, salicylic acid, steroid hormones, alcohol, Safeners (WO 97/06269), or it may also be advantageous to employ organ-specific promoters.
  • plant storage organ specific promoter refers to a promoter that preferentially, when compared to other plant tissues, directs gene transcription in a storage organ of a plant.
  • sink tissues of the plant such as the tuber of the potato plant, the fruit of tomato, or the seed of soybean, canola, cotton, Zea mays , wheat, rice, and barley
  • the promoters utilized in the present invention have relatively high expression in these specific tissues.
  • the promoter for /Z-conglycinin or other seed- specific promoters such as the napin, zein, linin and phaseolin promoters, can be used.
  • Root specific promoters may also be used.
  • An example of such a promoter is the promoter for the acid chitinase gene. Expression in root tissue could also be accomplished by utilizing the root specific subdomains of the CaMV 35S promoter that have been identified.
  • the plant storage organ specific promoter is a fruit specific promoter.
  • fruit specific promoter examples include, but are not limited to, the tomato polygalacturonase, E8 and Pds promoters, as well as the apple ACC oxidase promoter (for review, see Potenza et ah, 2004).
  • the promoter preferentially directs expression in the edible parts of the fruit, for example the pith of the fruit, relative to the skin of the fruit or the seeds within the fruit.
  • the inducible promoter is the Aspergillus nidulans ale system. Examples of inducible expression systems which can be used instead of the Aspergillus nidulans ale system are described in a review by Padidam (2003) and Corrado and Karali (2009).
  • the inducible promoter is a safener inducible promoter such as, for example, the maize ln2-l or ln.2-2 promoter (Hershey and Stoner, 1991), the safener inducible promoter is the maize GST-27 promoter (Jepson et ah, 1994), or the soybean GH2/4 promoter (Ulmasov et ah, 1995).
  • the inducible promoter is a senescence inducible promoter such as, for example, senescence-inducible promoter SAG (senescence associated gene) 12 and SAG 13 from Arabidopsis (Gan, 1995; Gan and Amasino, 1995) and LSC54 from Brassica napus (Buchanan-Wollaston, 1994).
  • SAG senescence-inducible promoter
  • SAG 13 senescence associated gene 12 and SAG 13 from Arabidopsis (Gan, 1995; Gan and Amasino, 1995) and LSC54 from Brassica napus (Buchanan-Wollaston, 1994).
  • SAG senescence-inducible promoter
  • SAG senescence associated gene 12 and SAG 13 from Arabidopsis
  • LSC54 from Brassica napus
  • ribulose biphosphate carboxylase For expression in vegetative tissue leaf-specific promoters, such as the ribulose biphosphate carboxylase (RBCS) promoters, can be used.
  • RBCS ribulose biphosphate carboxylase
  • the tomato RBCS1, RBCS2 and RBCS3A genes are expressed in leaves and light grown seedlings (Meier et al., 1997).
  • Another leaf-specific promoter is the light harvesting chlorophyll a/b binding protein gene promoter (see, Shiina et al., 1997).
  • the Arabidopsis thaliana myb-related gene promoter (Atmyb5) described by Li et al. (1996), is leaf- specific.
  • the Atmyb5 promoter is expressed in developing leaf trichomes, stipules, and epidermal cells on the margins of young rosette and cauline leaves, and in immature seeds.
  • a leaf promoter identified in maize by Busk et al. (1997), can also be used.
  • transgene is not expressed at high levels.
  • An example of a promoter which can be used in such circumstances is a truncated napin A promoter which retains the seed-specific expression pattern but with a reduced expression level (Tan et al., 2011).
  • the 5' non-translated leader sequence can be derived from the promoter selected to express the heterologous gene sequence of an RNA molecule of the present disclosure, or may be heterologous with respect to the coding region of the enzyme to be produced, and can be specifically modified if desired so as to increase translation of mRNA.
  • the 5' non-translated regions can also be obtained from plant viral RNAs (Tobacco mosaic vims, Tobacco etch vims, Maize dwarf mosaic vims, Alfalfa mosaic vims, among others), plant genes (wheat and maize chlorophyll a/b binding protein gene leader), or from a synthetic gene sequence.
  • the present invention is not limited to constructs wherein the non-translated region is derived from the 5' non-translated sequence that accompanies the promoter sequence.
  • the leader sequence could also be derived from an unrelated promoter or coding sequence.
  • Leader sequences useful in context of the present invention comprise the maize Hsp70 leader (US 5,362,865 and US 5,859,347), and the TMV omega element.
  • the termination of transcription is accomplished by a 3' non-translated DNA sequence operably linked in the expression vector to the RNA molecule of interest.
  • the 3' non-translated region of a recombinant DNA molecule contains a polyadenylation signal that functions in plants to cause the addition of adenylate nucleotides to the 3' end of the RNA.
  • the 3' non-translated region can be obtained from various genes that are expressed in plant cells.
  • the nopaline synthase 3' untranslated region, the 3' untranslated region from pea small subunit Rubisco gene, the 3' untranslated region from soybean 7S seed storage protein gene are commonly used in this capacity.
  • the 3' transcribed, non-translated regions containing the polyadenylate signal of Agrobacterium tumor-inducing (Ti) plasmid genes are also suitable.
  • the expression vector comprises a nucleic acid sequence as shown in SEQ ID NO: 150.
  • Transfer nucleic acids can be used to deliver an exogenous polynucleotide to a cell and comprise one, preferably two, border sequences and one or more RNA molecules of interest.
  • the transfer nucleic acid may or may not encode a selectable marker.
  • the transfer nucleic acid forms part of a binary vector in a bacterium, where the binary vector further comprises elements which allow replication of the vector in the bacterium, selection, or maintenance of bacterial cells containing the binary vector.
  • the transfer nucleic acid component of the binary vector is capable of integration into the genome of the plant cell or, for transient expression experiments, merely of expression in the cell.
  • extrachromosomal transfer nucleic acid refers to a nucleic acid molecule that is capable of being transferred from a bacterium such as Agrobacterium sp., to a plant cell such as a plant leaf cell.
  • An extrachromosomal transfer nucleic acid is a genetic element that is well-known as an element capable of being transferred, with the subsequent integration of a nucleotide sequence contained within its borders into the genome of the recipient cell.
  • a transfer nucleic acid is flanked, typically, by two "border” sequences, although in some instances a single border at one end can be used and the second end of the transferred nucleic acid is generated randomly in the transfer process.
  • RNA molecule of interest is typically positioned between the left border-like sequence and the right border-like sequence of a transfer nucleic acid.
  • the RNA molecule contained within the transfer nucleic acid may be operably linked to a variety of different promoter and terminator regulatory elements that facilitate its expression, that is, transcription and/or translation of the RNA molecule.
  • Transfer DNAs from Agrobacterium sp. such as Agrobacterium tumefaciens or Agrobacterium rhizogenes, and man made variants/mutants thereof are probably the best characterized examples of transfer nucleic acids.
  • P-DNA plant-DNA
  • P-DNA comprises T-DNA border-like sequences from plants.
  • T-DNA refers to a T-DNA of an Agrobacterium tumefaciens Ti plasmid or from an Agrobacterium rhizogenes Ri plasmid, or variants thereof which function for transfer of DNA into plant cells.
  • the T-DNA may comprise an entire T- DNA including both right and left border sequences, but need only comprise the minimal sequences required in cis for transfer, that is, the right T-DNA border sequence.
  • the T-DNAs of the invention have inserted into them, anywhere between the right and left border sequences (if present), the RNA molecule of interest.
  • sequences encoding factors required in trans for transfer of the T-DNA into a plant cell may be inserted into the T-DNA, or may be present on the same replicon as the T-DNA, or preferably are in trans on a compatible replicon in the Agrobacterium host.
  • Such "binary vector systems" are well known in the art.
  • P-DNA refers to a transfer nucleic acid isolated from a plant genome, or man made variants/mutants thereof, and comprises at each end, or at only one end, a T-DNA border-like sequence.
  • a "border" sequence of a transfer nucleic acid can be isolated from a selected organism such as a plant or bacterium, or be a man made variant/mutant thereof.
  • the border sequence promotes and facilitates the transfer of the RNA molecule to which it is linked and may facilitate its integration in the recipient cell genome.
  • a border-sequence is between 10-80 bp in length. Border sequences from T-DNA from Agrobacterium sp. are well known in the art and include those described in Lacroix et al. (2008).
  • Agrobacterium sp. Whilst traditionally only Agrobacterium sp. have been used to transfer genes to plants cells, there are now a large number of systems which have been identified/developed which act in a similar manner to Agrobacterium sp. Several non- Agrobacterium species have recently been genetically modified to be competent for gene transfer (Chung et al., 2006; Broothaerts et al., 2005). These include Rhizobium sp. NGR234, Sinorhizobium meliloti and Mezorhizobium loti.
  • the terms “transfection”, “transformation” and variations thereof are generally used interchangeably.
  • “Transfected” or “transformed” cells may have been manipulated to introduce the RNA molecule(s) of interest, or may be progeny cells derived therefrom.
  • the transfer nucleic acid comprises a nucleic acid sequence as shown in SEQ ID NO: 150.
  • the invention also provides a recombinant cell, for example, a recombinant plant cell, which is a host cell transformed with one or more RNA molecules or vectors defined herein, or combination thereof.
  • Suitable cells of the invention include any cell that can be transformed with an RNA molecule or recombinant vector according to the present disclosure.
  • the host cell is a plant cell.
  • the recombinant cell may be a cell in culture, a cell in vitro , or in an organism such as for example, a plant, or in an organ such as, for example, a seed or a leaf.
  • the cell is in a plant, more preferably in the seed of a plant.
  • Host cells into which the RNA molecules(s) are introduced can be either untransformed cells or cells that are already transformed with at least one nucleic acid. Such nucleic acids may be related to lipid synthesis, or unrelated.
  • Host cells of the present invention either can be endogenously (i.e., naturally) capable of expressing RNA molecule(s) defined herein, in which case the recombinant cell derived therefrom has an enhanced capability of producing the RNA molecule(s), or can be capable of producing said RNA molecule(s) only after being transformed with at least one RNA molecule defined herein.
  • the cell is a cell which is capable of being used for producing lipid.
  • a recombinant cell of the invention has an enhanced capacity to produce non-polar lipid such as TAG.
  • the plant cell is a seed cell, in particular, a cell in a cotyledon or endosperm of a seed.
  • the invention also provides a plant comprising one or more exogenous RNA molecules defined herein, a cell of according to the present disclosure, a vector according to the present disclosure, or a combination thereof.
  • plant when used as a noun refers to whole plants, whilst the term “part thereof” refers to plant organs (e.g., leaves, stems, roots, flowers, fruit), single cells (e.g., pollen), seed, seed parts such as an embryo, endosperm, scutellum or seed coat, plant tissue such as vascular tissue, plant cells and progeny of the same.
  • plant parts comprise plant cells.
  • the terms “in a plant” and “in the plant” in the context of a modification to the plant means that the modification has occurred in at least one part of the plant, including where the modification has occurred throughout the plant, and does not exclude where the modification occurs in only one or more but not all parts of the plant.
  • a tissue- specific promoter is said to be expressed “in a plant”, even though it might be expressed only in certain parts of the plant.
  • a transcription factor polypeptide that increases the expression of one or more glycolytic and/or fatty acid biosynthetic genes in the plant means that the increased expression occurs in at least a part of the plant.
  • the term "plant” is used in it broadest sense, including any organism in the Kingdom Plantae. It also includes red and brown algae as well as green algae. It includes, but is not limited to, any species of flowering plant, grass, crop or cereal (e.g., oilseed, maize, soybean), fodder or forage, fruit or vegetable plant, herb plant, woody plant or tree. It is not meant to limit a plant to any particular structure. It also refers to a unicellular plant (e.g., microalga).
  • the term "part thereof" in reference to a plant refers to a plant cell and progeny of same, a plurality of plant cells, a structure that is present at any stage of a plant's development, or a plant tissue.
  • plant tissue includes differentiated and undifferentiated tissues of plants including those present in leaves, stems, flowers, fruits, nuts, roots, seed, seed coat, embryos.
  • plant tissue includes differentiated and undifferentiated tissues of plants including those present in leaves, stems, flowers, fruits, nuts, roots, seed, for example, embryonic tissue, endosperm, dermal tissue (e.g., epidermis, periderm), vascular tissue (e.g., xylem, phloem), or ground tissue (comprising parenchyma, collenchyma, and/or sclerenchyma cells), as well as cells in culture (e.g., single cells, protoplasts, callus, embryos, etc.).
  • Plant tissue may be in planta, in organ culture, tissue culture, or cell culture.
  • a “seedling consists” refers to the stage of plant growth spanning emergence from the seed up until the formation of the first true leaves.
  • the seedling comprises of three main parts: the radicle (embryonic root), the hypocotyl (embryonic shoot), and the cotyledon(s).
  • 18:3 and 16:3 fatty acids are found within the glycolipids of different plant species. This is used to distinguish between 18:3 plants whose fatty acids with 3 double bonds are generally always Cis atoms long and the 16:3 plants that contain both C ⁇ e- and C is- fatty acids.
  • 18:3 chloroplasts enzymic activities catalyzing the conversion of phosphatidate to diacylglycerol and of diacyiglycerol to monogalactosyl diacylglycerol (MGD) are significantly less active than in 16:3 chloroplasts.
  • chloroplasts synthesize stearoyl-ACP2 in the stroma, introduce the first double bond into the saturated hydrocarbon chain, and then hydrolyze the thioester. Released oleate is exported across chloroplast envelopes into membranes of the eucaryotic part of the cell, probably the endoplasmic reticulum, where it is incorporated into PC.
  • PC-linked oleoyl groups are desaturated in these membranes and subsequently move back into the chloroplast.
  • the MGD-linked acyl groups are substrates for the introduction of the third double bond to yield MGD with two linolenoyl residues.
  • This galactolipid is characteristic of 18:3 plants such as Asteraceae and Fabaceae, for example.
  • 16:3 plants which are represented, for example, by members of Apiaceae and Brassicaceae
  • two pathways operate in parallel to provide thylakoids with MGD.
  • the cooperative 'eucaryotic' sequence is supplemented to various extents by a 'procaryotic' pathway. Its reactions are confined to the chloroplast and result in a typical arrangement of acyl groups as well as their complete desaturation once they are esterified to MGD.
  • Procaryotic DAG backbones carry 06:0 and its desaturation products at C-2 from which position 08: fatty acids are excluded.
  • the C-l position is occupied by 08 fatty acids and to a small extent by 06 groups.
  • the similarity in DAG backbones of lipids from blue-green algae with those synthesized by the chloroplast-confmed pathway in 16:3 plants suggests a phylogenetic relation and justifies the term procaryotic.
  • the term "vegetative tissue” or “vegetative plant part” is any plant tissue, organ or part other than organs for sexual reproduction of plants.
  • the organs for sexual reproduction of plants are specifically seed bearing organs, flowers, pollen, fruits and seeds.
  • Vegetative tissues and parts include at least plant leaves, stems (including bolts and tillers but excluding the heads), tubers and roots, but excludes flowers, pollen, seed including the seed coat, embryo and endosperm, fruit including mesocarp tissue, seed-bearing pods and seed-bearing heads.
  • the vegetative part of the plant is an aerial plant part.
  • the vegetative plant part is a green part such as a leaf or stem.
  • transgenic plant refers to a plant that contains a transgene not found in a wild-type plant of the same species, variety or cultivar.
  • Transgenic plants as defined in the context of the present invention include plants and their progeny which have been genetically modified using recombinant techniques to cause production of at least one polypeptide defined herein in the desired plant or part thereof.
  • Transgenic plant parts has a corresponding meaning.
  • seed and “grain” are used interchangeably herein.
  • "Grain” refers to mature grain such as harvested grain or grain which is still on a plant but ready for harvesting, but can also refer to grain after imbibition or germination, according to the context. Mature grain commonly has a moisture content of less than about 18%. In a preferred embodiment, the moisture content of the grain is at a level which is generally regarded as safe for storage, preferably between 5% and 15%, between 6% and 8%, between 8% and 10%, or between 10% and 15%.
  • "Developing seed” as used herein refers to a seed prior to maturity, typically found in the reproductive structures of the plant after fertilisation or anthesis, but can also refer to such seeds prior to maturity which are isolated from a plant. Mature seed commonly has a moisture content of less than about 12%.
  • plant storage organ refers to a part of a plant specialized to store energy in the form of for example, proteins, carbohydrates, lipid.
  • plant storage organs are seed, fruit, tuberous roots, and tubers.
  • a preferred plant storage organ of the invention is seed.
  • the term "phenotypically normal” refers to a genetically modified plant or part thereof, for example a transgenic plant, or a storage organ such as a seed, tuber or fruit of the invention not having a significantly reduced ability to grow and reproduce when compared to an unmodified plant or part thereof.
  • the biomass, growth rate, germination rate, storage organ size, seed size and/or the number of viable seeds produced is not less than 90% of that of a plant lacking said recombinant polynucleotide when grown under identical conditions. This term does not encompass features of the plant which may be different to the wild-type plant but which do not affect the usefulness of the plant for commercial purposes such as, for example, a ballerina phenotype of seedling leaves.
  • the genetically modified plant or part thereof which is phenotypically normal comprises a recombinant polynucleotide encoding a silencing suppressor operably linked to a plant storage organ specific promoter and has an ability to grow or reproduce which is essentially the same as a corresponding plant or part thereof not comprising said polynucleotide.
  • Plants provided by or contemplated for use in the practice of the present invention include both monocotyledons and dicotyledons.
  • the plants of the present invention are crop plants (for example, cereals and pulses, maize, wheat, potatoes, rice, sorghum, millet, cassava, barley) or legumes such as soybean, beans or peas.
  • the plants may be grown for production of edible roots, tubers, leaves, stems, flowers or fruit.
  • the plants may be vegetable plants whose vegetative parts are used as food.
  • the plants of the invention may be: Acrocomia aculeata (macauba palm), Arabidopsis thaliana, Aracinis hypogaea (peanut), Astrocaryum murumuru (murumuru), Astrocaryum vulgare (tucuma), Attalea geraensis (Indaia-rateiro), Attalea humilis (American oil palm), Attalea oleifera (andaia), Attalea phalerata (uricuri), Attalea speciosa (babassu), Avena sativa (oats), Beta vulgaris (sugar beet), Brassica sp.
  • Brassica carinata such as Brassica carinata, Brassica juncea, Brassica napobrassica, Brassica napus (canola), Camelina sativa (false flax), Cannabis sativa (hemp), Carthamus tinctorius (safflower), Caryocar brasiliense (pequi), Cocos nucifera (Coconut), Crambe abyssinica (Abyssinian kale), Cucumis melo (melon), Elaeis guineensis (African palm), Glycine max (soybean), Gossypium hirsutum (cotton), Helianthus sp.
  • Brassica carinata such as Brassica carinata, Brassica juncea, Brassica napobrassica, Brassica napus (canola), Camelina sativa (false flax), Cannabis sativa (hemp), Carthamus tinctorius (safflower),
  • Lemna sp. such as Helianthus annuus (sunflower), Hordeum vulgare (barley), Jatropha curcas (physic nut), Joannesia princeps (arara nut-tree), Lemna sp. (duckweed) such as Lemna aequinoctialis, Lemna disperma, Lemna ecuadoriensis, Lemna gibba (swollen duckweed), Lemna japonica, Lemna minor, Lemna minuta, Lemna obscura, Lemna paucicostata, Lemna perpusilla, Lemna tenera, Lemna trisulca, Lemna turionifera, Lemna valdiviana, Lemna yachesis, Licania rigida (oiticica), Linum usitatissimum (flax), Lupinus angustifolius (lupin), Mauritia
  • Nicotiana sp. such as Miscanthus x giganteus and Miscanthus sinensis, Nicotiana sp. (tabacco) such as Nicotiana tabacum or Nicotiana benthamiana, Oenocarpus bacaba (bacaba-do-azeite), Oenocarpus bataua (pataua), Oenocarpus distichus (bacaba-de-leque), Oryza sp.
  • rice such as Oryza sativa and Oryza glaberrima, Panicum virgatum (switchgrass), Paraqueiba paraensis (mari), Persea amencana (avocado), Pongamia pinnata (Indian beech), Populus trichocarpa, Ricinus communis (castor), Saccharum sp. (sugarcane), Sesamum indicum (sesame), Solanum tuberosum (potato), Sorghum sp.
  • Triticum sp. such as Sorghum bicolor, Sorghum vulgare, Theobroma grandiforum (cupuassu), Trifolium sp., Trithrinax brasiliensis (Brazilian needle palm), Triticum sp. (wheat) such as Triticum aestivum, Zea mays (corn), alfalfa ( Medicago sativa), rye ( Secale cerale), sweet potato ( Lopmoea batatus), cassava ( Manihot esculenta), coffee ( Cofea spp.), pineapple (Anana comosus ), citris tree (Citrus spp.), cocoa ( Theobroma cacao), tea ( Camellia senensis), banana ( Musa spp.), avocado ( Persea americana), fig ( Ficus casica), guava ( Psidium guajava ), mango ( Mangifer indica), olive ( Ole
  • C4 grasses such as, in addition to those mentioned above, Andropogon gerardi, Bouteloua curtipendula, B. gracilis, Buchloe dactyloides, Schizachyrium scoparium, Sorghastrum nutans, Sporobolus cryptandrus, C3 grasses such as Elymus canadensis, the legumes Lespedeza capitata and Petalostemum villosum, the forb Aster azureus, and woody plants such as Quercus ellipsoidalis and Q. macrocarpa.
  • Other preferred plants include C3 grasses.
  • the plant is an angiosperm.
  • the plant is an oilseed plant, preferably an oilseed crop plant.
  • an "oilseed plant” is a plant species used for the commercial production of lipid from the seeds of the plant.
  • the oilseed plant may be, for example, oil- seed rape (such as canola), maize, sunflower, safflower, soybean, sorghum, flax (linseed) or sugar beet.
  • the oilseed plant may be other Brassicas, cotton, peanut, poppy, rutabaga, mustard, castor bean, sesame, safflower, Jatropha curcas or nut producing plants.
  • the plant may produce high levels of lipid in its fruit such as olive, oil palm or coconut.
  • Horticultural plants to which the present invention may be applied are lettuce, endive, or vegetable Brassicas including cabbage, broccoli, or cauliflower.
  • the present invention may be applied in tobacco, cucurbits, carrot, strawberry, tomato, or pepper.
  • the plant is a non-transgenic plant.
  • the transgenic plant is homozygous for each and every gene that has been introduced (transgene) so that its progeny do not segregate for the desired phenotype.
  • the transgenic plant may also be heterozygous for the introduced transgene(s), preferably uniformly heterozygous for the transgene such as for example, in FI progeny which have been grown from hybrid seed. Such plants may provide advantages such as hybrid vigour, well known in the art.
  • RNA molecules disclosed herein may be stably introduced to above referenced host cells and/or plants.
  • an example of the present disclosure encompasses an above referenced plant stably transformed with an RNA molecule disclosed herein.
  • the terms "stably transforming", “stably transformed” and variations thereof refer to the integration of the RNA molecule or a nucleic acid encoding the same into the genome of the cell such that they are transferred to progeny cells during cell division without the need for positively selecting for their presence.
  • Stable transformants, or progeny thereof can be identified by any means known in the art such as Southern blots on chromosomal DNA, or in situ hybridization of genomic DNA, enabling their selection.
  • Transgenic plants can be produced using techniques known in the art, such as those generally described in Slater et ah, Plant Biotechnology - The Genetic Manipulation of Plants, Oxford University Press (2003), and Christou and Klee, Handbook of Plant Biotechnology, John Wiley and Sons (2004).
  • plants may be transformed by topically applying an RNA molecule according to the present disclosure to the plant or a part thereof.
  • the RNA molecule may be provided as a formulation with a suitable carrier and sprayed, dusted or otherwise applied to the surface of a plant or part thereof.
  • the methods of the present disclosure encompass introducing an RNA molecule disclosed herein to a plant, the method comprising topically applying a composition comprising the RNA molecule to the plant or a part thereof.
  • Agrobacterium- mediated transfer is a widely applicable system for introducing genes into plant cells because DNA can be introduced into cells in whole plant tissues, plant organs, or explants in tissue culture, for either transient expression, or for stable integration of the DNA in the plant cell genome.
  • floral-dip (in planta ) methods may be used.
  • the use of Agrobacterium- mediated plant integrating vectors to introduce DNA into plant cells is well known in the art.
  • the region of DNA to be transferred is defined by the border sequences, and the intervening DNA (T-DNA) is usually inserted into the plant genome. It is the method of choice because of the facile and defined nature of the gene transfer.
  • Acceleration methods include for example, microprojectile bombardment and the like.
  • One example of a method for delivering transforming nucleic acid molecules to plant cells is microprojectile bombardment. This method has been reviewed by Yang et ah, Particle Bombardment Technology for Gene Transfer, Oxford Press, Oxford, England (1994).
  • Non-biological particles that may be coated with nucleic acids and delivered into cells, for example of immature embryos, by a propelling force.
  • Exemplary particles include those comprised of tungsten, gold, platinum, and the like.
  • plastids can be stably transformed.
  • Methods disclosed for plastid transformation in higher plants include particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination (US 5,451,513, US 5,545,818, US 5,877,402, US 5,932479, and WO 99/05265).
  • Other methods of cell transformation can also be used and include but are not limited to the introduction of DNA into plants by direct DNA transfer into pollen, by direct injection of DNA into reproductive organs of a plant, or by direct injection of DNA into the cells of immature embryos followed by the rehydration of desiccated embryos.
  • This regeneration and growth process typically includes the steps of selection of transformed cells, culturing those individualized cells through the usual stages of embryonic development through the rooted plantlet stage. Transgenic embryos and seeds are similarly regenerated. The resulting transgenic rooted shoots are thereafter planted in an appropriate plant growth medium such as soil.
  • the development or regeneration of plants containing the foreign, exogenous gene is well known in the art.
  • the regenerated plants are self-pollinated to provide homozygous transgenic plants. Otherwise, pollen obtained from the regenerated plants is crossed to seed-grown plants of agronomically important lines. Conversely, pollen from plants of these important lines is used to pollinate regenerated plants.
  • a transgenic plant of the present invention containing a desired polynucleotide is cultivated using methods well known to one skilled in the art.
  • transgenic plants may be grown to produce plant tissues or parts having the desired phenotype.
  • the plant tissue or plant parts may be harvested, and/or the seed collected.
  • the seed may serve as a source for growing additional plants with tissues or parts having the desired characteristics.
  • the vegetative plant parts are harvested at a time when the yield of non-polar lipids are at their highest.
  • the vegetative plant parts are harvested about at the time of flowering, or after flowering has initiated.
  • the plant parts are harvested at about the time senescence begins, usually indicated by yellowing and drying of leaves.
  • Transgenic plants formed using Agrobacterium or other transformation methods typically contain a single genetic locus on one chromosome. Such transgenic plants can be referred to as being hemizygous for the added gene(s). More preferred is a transgenic plant that is homozygous for the added gene(s), that is, a transgenic plant that contains two added genes, one gene at the same locus on each chromosome of a chromosome pair.
  • a homozygous transgenic plant can be obtained by self-fertilising a hemizygous transgenic plant, germinating some of the seed produced and analysing the resulting plants for the gene of interest.
  • transgenic plants that contain two independently segregating exogenous genes or loci can also be crossed (mated) to produce offspring that contain both sets of genes or loci.
  • Selfing of appropriate FI progeny can produce plants that are homozygous for both exogenous genes or loci.
  • Back-crossing to a parental plant and out-crossing with a non-transgenic plant are also contemplated, as is vegetative propagation.
  • a transgenic plant can be crossed with a second plant comprising a genetic modification such as a mutant gene and progeny containing both of the transgene and the genetic modification identified. Descriptions of other breeding methods that are commonly used for different traits and crops can be found in Fehr, In: Breeding Methods for Cultivar Development, Wilcox J. ed., American Society of Agronomy, Madison Wis. (1987).
  • RNA molecules of the invention can be provided as various formulations.
  • RNA molecules may be in the form of a solid, ointment, gel, cream, powder, paste, suspension, colloid, foam or aerosol.
  • Solid forms may include dusts, powders, granules, pellets, pills, pastilles, tablets, filled films (including seed coatings) and the like, which may be water-dispersible ("wettable").
  • the composition is in the form of a concentrate.
  • RNA molecules may be provided as a topical formulation.
  • the formulation stabilises the RNA molecule in formulation and/or in-vivo.
  • RNA molecules of the invention may be provided in a lipid formulation.
  • the formulation comprises a transfection promoting agent.
  • transfection promoting agent refers to a composition added to the RNA molecule for enhancing the uptake into a cell including, but not limited to, a plant cellor a fungal cell. Any transfection promoting agent known in the art to be suitable for transfecting cells may be used.
  • Examples include cationic lipid such as one or more of DOTMA (N-[l-(2.3-dioleoyloxy)-propyl]-N,N,N-trimethyl ammonium chloride), DOTAP (l,2-bis(oleoyloxy)-3-3-(trimethylammonium)propane), DMRIE (l,2-dimyristyloxypropyl-3 -dimethyl -hydroxy ethyl ammonium bromide), DDAB (dimethyl dioctadecyl ammonium bromide) lipospermines, specifically DOSPA (2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-l- propanamin- ium trifluoro-acetate) and DOSPER (l,3-dioleoyloxy-2-(6carboxy spermyl)-propyl-amid, and the di- and t
  • Cationic lipids are optionally combined with non-cationic lipids, particularly neutral lipids, for example lipids such as DOPE (dioleoylphosphatidylethanolamine), DPhPE (diphytanoylphosphatidylethanolamine) or cholesterol.
  • DOPE dioleoylphosphatidylethanolamine
  • DPhPE diphytanoylphosphatidylethanolamine
  • suitable commercially available transfection reagents include Lipofectamine (Life Technologies) and Lipofectamine 2000 (Life Technologies).
  • RNA molecules of the invention can be incorporated into formulations suitable for application to a field.
  • the field comprises plants.
  • Suitable plants include crop plants (for example, cereals and pulses, maize, wheat, potatoes, tapioca, rice, sorghum, soybean millet, cassava, barley, or pea), or legumes.
  • the plants may be grown for production of edible roots, tubers, leaves, stems, flowers or fruit.
  • the crop plant is a cereal plant.
  • cereal plants include, but are not limited to, wheat, barley, sorghum oats, and rye.
  • the RNA molecule may be formulated for administration to the plant, or to any part of the plant, in any suitable way.
  • the composition may be formulated for administration to the leaves, stem, roots, fruit vegetables, grains and/or pulses of the plant.
  • the RNA molecule is formulated for administration to the leaves of the plant, and is sprayable onto the leaves of the plant.
  • RNA molecules of the invention may be formulated with a variety of other agents.
  • agents comprise one or more of suspension agents, agglomeration agents, bases, buffers, bittering agents, fragrances, preservatives, propellants, thixotropic agents, anti-freezing agents, and colouring agents.
  • RNA molecule formulations can further comprise an insecticide, a pesticide, a fungicide, an antibiotic, an insect repellent, an anti-parasitic agent, an anti- viral agent, or a nematicide.
  • RNA molecules according to the present disclosure can be provided in a kit or pack.
  • RNA molecules disclosed herein may be packaged in a suitable container with written instructions for producing an above referenced cell or plant.
  • the RNA molecules according to the present disclosure can be delivered to plants, plant cells or plant parts, preferably to seed that will be used to produce plants, to modulate flowering. Such uses involve delivering RNA molecules according to the present disclosure using various methods such as those described above for delivering RNA molecules.
  • plants disclosed herein can be modified to express RNA molecules according to the present disclosure.
  • RNA molecules can be sprayed onto plants as required.
  • RNA molecules can be sprayed onto a crop to promote flowering in the crop.
  • the RNA molecules according to the present disclosure can be delivered to plants to modulate vernalization.
  • Exemplary crops include cotton, maize, tomato, chickpea, pigeon pea, alfalfa, rice, sorghum and cowpea.
  • Other exemplary crops include corn, canola, cotton, soybean, wheat, barley, rice, legume, Medicago truncatula, sugarbeet or rye.
  • Further examples of suitable plants and crops are discussed throughout the present disclosure.
  • the methods of the present disclosure can be used to modulate flowering in plants such as Arabidopsis, com, canola, cotton, soybean, alfalfa, lettuce, wheat, barley, rice, legume, Medicago truncatula , sugarbeet or rye.
  • the methods of the present disclosure can be used to modulate flowering in plants such as Arabidopsis, com, canola, cotton, soybean, wheat, bareley, rice, legume, Medicago truncatula, sugarbeet or rye.
  • the plant can be sugarbeet.
  • the plant is wheat or barley.
  • the methods of the present disclosure are used to direct early flowering in plants such as Arabidopsis, corn, canola, cotton, soybean, alfalfa, lettuce, wheat, barley, rice, legume, Medicago truncatula, sugarbeet or rye.
  • the methods of the present disclosure are used to direct early flowering in plants such as Arabidopsis, com, canola, cotton, soybean, wheat, bareley, rice, legume, Medicago truncatula, sugarbeet or rye.
  • early flowering can be directed in sugarbeet.
  • the early flowering is directed in wheat or barley.
  • the methods of the present disclosure can be used to modulate flowering in grass such as turfgrasses.
  • the methods of the present disclosure are used to direct late flowering in a grass.
  • the late flowering is directed in turfgrasses.
  • RNA molecules of the disclosure are delivered to genetically unmodified plants.
  • RNA molecules of the disclosure when delivered and/or expressed in a plant can have a wide range of desired properties which influence, for example, an agronomic trait such as early flowering.
  • the plants produce increased levels of enzymes for oil production in plants such as Brassicas, for example oilseed rape or sunflower, safflower, flax, cotton, soybean or maize; enzymes involved in starch synthesis in plants such as potato, maize, and cereals such as wheat barley or rice; enzymes which synthesize, or proteins which are themselves, natural medicaments, such as pharmaceuticals or veterinary products.
  • plants such as Brassicas, for example oilseed rape or sunflower, safflower, flax, cotton, soybean or maize
  • enzymes involved in starch synthesis in plants such as potato, maize, and cereals such as wheat barley or rice
  • enzymes which synthesize, or proteins which are themselves, natural medicaments, such as pharmaceuticals or veterinary products are themselves, natural medicaments, such as pharmaceuticals or veterinary products.
  • exemplary physical or phenotypic characteristics of plants produced from the plant cells or seeds contacted with the RNA molecules of the invention may be affected in addition to the modulated flowering time phenotype, such as reduced chlorophyll content, stem elongation, advanced or retarded senescence, and increase or reduction of apical dominance which may result in an altered plant architecture, each of which are different from the plant phenotype when grown in the absence of the contact with the RNA molecules.
  • these phenotypes if deleterious, are advantageously reduced or absent in the subsequent generation of plants which may be used for producing grain, fruits, pods or vegetative parts such as leaves, stems, fibre, tubers or beets.
  • the RNA molecules of the invention can be used in the previous generation to induce earlier flowering for seed production, in an otherwise later-flowering variety in the absence of treatment with the RNA molecules.
  • sugarbeet which stores sugar in the beets in the vegetative state but mobilises that sugar at the onset of flowering, leading to reduction in sugar content in the beets. Since hybrid seed stock is sown for the cultivation of sugar beets it must be ensured that the parent plants still flower in order to produce the seed stock
  • the 5’ end of the resultant construct was preceded with a promoter such as a T7 or SP6 RNA polymerase promoter and the 3’ end engineered to include a restriction enzyme cleavage site to allow for termination of transcription in vitro.
  • promoter and terminator sequences were incorporated to facilitate expression as a transgene, for example using an inducible promoter.
  • the double- stranded region and loop sequence lengths can be varied.
  • the constructs were made using standard cloning methods or ordered from commercial service providers.
  • RNA polymerase resulted in the 5’ and 3’ arms of the ledRNAi transcript annealing to the central target sequence, the molecule comprising a central stem or double-stranded region with a single nick and terminal loops.
  • the central sequence can be orientated in sense or antisense orientation relative to the promoter ( Figure 1A, IB respectively).
  • RNA synthesis was achieved using RNA polymerase according to the manufacturer’s instructions.
  • the ledRNA was resuspended in annealing buffer (25 mM Tris-HCL, pH 8.0, 10 mM MgCh) using DEPC-treated water to inactivate any traces of RNAse.
  • the yield and integrity of the RNA produced by this method was determined by nano-drop analysis and gel electrophoresis (Figure 2), respectively.
  • RNA transcription with Cy3 labelling the ribonucleotide (rNTP) mix contained lOmM each of ATP, GTP, CTP, 1.625mM UTP and 8.74mM Cy3-UTP.
  • the transcription reactions were incubated at 37°C for 2.5 hr.
  • the transcription reactions 160pl were the transferred to Eppendorf tubes, 17.7pl turbo DNase buffer and Im ⁇ turbo DNA added, and incubated at 37°C for 10 minutes to digest the DNA.
  • 17.7m1 Turbo DNAse inactivation solution was added, mixed and incubated at room temperature for 5 min. The mixture was centifuged for 2 min and the supernatant transferred to a new RNAse free Eppendorf tube.
  • RNA samples of 1.5pl of each transcription reaction were electrophoresed on gels to test the quality of the RNA product. Generally, one RNA band was observed of 500 bp to lOOObp in size depending on the construct.
  • the RNA was precipitated by adding to each tube: 88.5m1 7.5M Ammonium acetate and 665m1 cold 100% ethanol. The tubes were cooled to -20°C for several hours or overnight, then centrifuged at 4°C for 30 min. The supernatant was removed carefully and the pellet of RNA washed with 1ml 70% ethanol (made with nuclease free water) at -20°C and centifuged. The pellet was dried and the purified RNA resuspended in 50m1 lx RNAi annealing buffer. The RNA concentration was measured using nanodrop method and stored at -80°C until used.
  • a typical ledRNA molecule comprises a sense sequence which can be considered to be two adjacent sense sequences, covalently linked and having identity to the target RNA, an antisense sequence which is complementary to the sense sequence and which is divided into two regions, and two loops that separate the sense from the antisense sequences.
  • a DNA construct which encodes this form of ledRNA therefore comprises, in 5’ to 3’ order, a promoter for transcription of the ledRNA coding region, a first antisense region having complementarity with a region towards the 5’ end of the target RNA, a first loop sequence, the sense sequence, a second loop sequence, then the second antisense region having complementarity with a region towards the 3’ end of the target RNA, and finally a means to terminate transcription.
  • the two antisense sequences flanked the sense sequence and loop sequences. When transcribed, the two regions of antisense sequence anneal with the sense sequence, forming a dsRNA stem with two flanking loops.
  • a DNA construct which encodes this second form of ledRNA therefore comprises, in 5’ to 3’ order, a promoter for transcription of the ledRNA coding region, a first sense region having identity with a region towards the 3’ end of the target RNA, a first loop sequence, the antisense sequence, a second loop sequence, then the second sense region having identity towards the 5’ end of the target RNA, and finally a means to terminate transcription.
  • the two sense sequences flanked the antisense sequence and loop sequences.
  • these ledRNA structures would be more resistant to exonucleases than an open- ended dsRNA formed between single- stranded sense and antisense RNAs and not having loops, and also compared to a hairpin RNA having only a single loop.
  • the inventors conceived that a loop at both ends of the dsRNA stem would allow Dicer to access both ends efficiently, thereby enhancing processing of the dsRNA into sRNAs and silencing efficiency.
  • a genetic construct was made for in vitro transcription using T7 or SP6 RNA polymerase to form ledRNAs targeting genes encoding GFP or GUS.
  • the ledGFP construct comprised the following regions in order: the first half of antisense sequence corresponded to nucleotides 358 to 131 of the GFP coding sequence (CDS) (SEQ ID NO:7), the first antisense loop corresponded to nucleotides 130 to 1 of GFP CDS, the sense sequence corresponded of nucleotides 131 to 591 of GFP CDS, the second antisense loop corresponding to nucleotides 731 to 592 of GFP CDS, and the second half of the antisense sequence corresponded to nucleotides 591 to 359 of the GFP CDS.
  • CDS GFP coding sequence
  • the ledGUS construct comprised the following regions in order: the first half of antisense sequence corresponded to nucleotides 609 to 357 of GUS CDS (SEQ ID NO:8); the first antisense loop corresponded to nucleotides 356 to 197 of GUS CDS, the sense sequence corresponded to nucleotides 357 to 860 of GUS CDS, the second antisense loop corresponding to nucleotides 1029 to 861 of GUS CDS; and the second half of antisense sequence corresponded to nucleotides 861 to 610 of GUS CDS.
  • the same target sequence corresponding to nucleotides 357 to 860 of GUS CDS was ligated between the T7 and SP6 promoters in pGEM-T Easy vector.
  • the sense and antisense strands were transcribed separately with T7 or SP6 polymerases, respectively, and annealed in annealing buffer after mixing the transcripts and heating the mixture to denature the RNA strands.
  • ledRNA The ability of ledRNA to form dsRNA structures was compared with open- ended dsRNA (i.e no loops, formed by annealing of separate single- stranded sense and antisense RNA) and long hpRNA.
  • ledRNA, long hpRNA, and the mixture of sense and antisense RNA were denatured by boiling and allowed to anneal in annealing buffer (250mM Tris-HCL, pH 8.0 and lOOmM MgC12), and then subjected to electrophoresis in a 1.0% agarose gel under non-denaturing conditions.
  • annealing buffer 250mM Tris-HCL, pH 8.0 and lOOmM MgC12
  • both the GUS ledRNA and the GFP ledRNA gave a dominant RNA band of the mobility expected for a double-stranded molecule, indicating the formation of the predicted ledRNA structure. This was in contrast to the mixture of sense and antisense RNA, which showed only a weak band for a dsRNA, indicating that most of the sense and antisense RNAs were not readily annealed to each other to form dsRNA.
  • the hairpin RNA samples gave two prominent bands, indicating that only part of the transcript formed the predicted hairpin RNA structure. Thus, ledRNA was the most efficient in forming the predicted dsRNA structure.
  • the ability of the ledRNAs to induce RNAi after topical delivery was tested in Nicotiana benthamiana and Nicotiana tabacum plants expressing a GFP or GUS reporter gene, respectively.
  • the sequences of the GFP and GUS target sequences and of the ledRNA encoding constructs are shown in SEQ ID NOs: 7, 8, 4 and 5, respectively.
  • the ribonucleotide sequence of the encoded RNA molecules are provided as SEQ ID NO’s 1 (GFP ledRNA) and 2 (GUS ledRNA).
  • ledRNA at a concentration of 75-100 pg/ml, in 25 mM Tris-HCL, pH 8.0, 10 mM MgCh and Silwet 77 (0.05%), was applied to the adaxial surface of leaves using a soft paint brush. At 6 hours and 3 days following ledRNA application, leaf samples were taken for the analysis of targeted gene silencing.
  • a ledRNA was designed to target an mRNA encoded by an endogenous gene, namely the FAD2.1 gene of N. benthamiana.
  • the sequence of the target FAD2.1 mRNA and of the ledFAD2.1 encoding construct are shown in SEQ ID NOs: 9 and 6, respectively.
  • the ribonucleotide sequence of the encoded RNA molecule is provided as SEQ ID NOG (N. benthamiana FAD2.1 ledRNA).
  • the FAD2.1 ledRNA construct was comprised of the following: the first half of antisense sequence corresponding to nucleotides 678 to 379 of FAD2.1 CDS (Nibenl01Scf09417g01008.1); the first antisense loop corresponding to nt. 378 to 242 of FAD2.1 CDS; the sense sequence corresponding of nt. 379 to 979; the second antisense loop corresponding to nt 1115 to 980; and the second half of antisense sequence corresponding to nt 979 to nt 679 of FAD2.1 CDS.
  • the ledGUS RNA from the previous example was used in parallel as a negative control.
  • target gene silencing was assayed for both the level of FAD2.1 mRNA and the accumulation 08:1 fatty acid ( Figure 5).
  • the level of activity of a related gene, FAD2.2 was also assayed.
  • For each sample approximately 3 pg of total RNA was DNase treated and reverse transcribed at 50 °C for 50 minutes using oligo dT primers. The reactions were terminated at 85 °C for 5 minutes and diluted to 120 pi with water.
  • the FAD2.1 mRNA was reduced significantly, to a level which was barley detectable in leaf tissues treated with the ledRNA at the 2, 4 and 10 hour time points (Figure 5). In this experiment, it was unclear why the level of FAD2.1 mRNA was not reduced as much at the 6 hour time point. In the repeated experiment shown in Figure 6, strong FAD2.1 downregulation occurred at both 6 and 24 hrs, particularly at the 24 hr time point. The related FAD2.2 gene, with sequence homology to FAD2.1, also showed downregulation at the 2 and 4 hour time points by the ledRNA (Figure 5).
  • Reporter genes such as the gene encoding the enzyme B-glucuronidase (GUS) provide a simple and convenient assay system that can be used to measure gene silencing efficiency in a eukaryotic cell including in plant cells (Jefferson et ah, 1987).
  • the inventors therefore designed, produced and tested some modified hairpin RNAs for their ability to reduce the expression of a GUS gene as a target gene, using a gene- delivered approach to provide the hairpin RNAs to the cells, and compared the modified hairpins to a conventional hairpin RNA.
  • the conventional hairpin RNA used as the control in the experiment had a double- stranded region of 200 contiguous basepairs in length in which all of the basepairs were canonical basepairs, i.e. G:C and A:U basepairs without any G:U basepairs, and without any non-basepaired nucleotides (mismatches) in the double- stranded region, targeting the same 200nt region of the GUS mRNA molecule as the modified hairpin RNAs.
  • the sense and antisense sequences that formed the double- stranded region were covalently linked by a spacer sequence included a PDK intron (Helliwell et ah, 2005; Smith et ah, 2000), providing for an RNA loop of 39 or 45 nucleotides in length (depending on the cloning strategy used) after splicing of the intron from the primary transcript.
  • the DNA fragment used for the antisense sequence was flanked by XhoI-BamHI restriction sites at the 5’ end and HindHI-Kpnl restriction sites at the 3’ end for easy cloning into an expression cassette, and each sense sequence was flanked by Xhol and Kpnl restriction sites.
  • each hairpin RNA both for the control hairpin and the modified hairpins, included an antisense sequence of 200 nucleotides which was fully complementary to a wild-type GUS sequence from within the protein coding region.
  • This antisense sequence corresponding to nucleotides 13-212 of SEQ ID NO: 10, was the complement of nucleotides 804-1003 of the GUS open reading frame (ORF) (cDNA sequence provided as SEQ ID NO:8).
  • ORF GUS open reading frame
  • the length of 200 nucleotides for the sense and antisense sequences was chosen as small enough to be reasonably convenient for synthesis of the DNA fragments using synthetic oligonucleotides, but also long enough to provide multiple sRNA molecules upon processing by Dicer. Being part of an ORF, the sequence was unlikely to contain cryptic splice sites or transcription termination sites.
  • the 200 bp GUS ORF sequence was PCR-amplified using the oligonucleotide primer pair GUS-WT-F (SEQ ID NO:52) and GUS-WT-R (SEQ ID NO:53), containing Xhol and BamHL sites or HindHI and Kpnl sites, respectively, to introduce these restriction enzyme sites 5’ and 3’ of the GUS sequence.
  • the amplified fragment was inserted into the vector pGEM-T Easy and the correct nucleotide sequence confirmed by sequencing.
  • the GUS fragment was excised by digestion with BamHI and HindHI and inserted into the BamHUHindHI site of pKannibal (Helliwell and Waterhouse, 2005), which inserted the GUS sequence in the antisense orientation relative to the operably linked CaMV e35S promoter (Grave, 1992) and ocs gene polyadenylation/transcription terminator (Ocs-T).
  • the resultant vector was designated pMBW606 and contained, in order 5’ to 3’, a 35S::PDK Intron::antisense GUS::Ocs-T expression cassette. This vector was the intermediate vector used as the base vector for assembling four hpRNA constructs.
  • the 200 bp GUS PCR fragment was excised from the pGEM-T Easy plasmid with Xhol and Kpnl, and inserted into the Xhol! Kpnl sites between the 35S promoter and the PDK intron in pMBW606.
  • This cassette was excised by digestion with Not I and inserted into the Not! site of pART27 (Gleave, 1992), resulting in the vector designated hpGUS[wt], encoding the canonically basepaired hairpin RNA targeting the GUS mRNA.
  • this hairpin When self-annealed by hybridisation of the 200nt sense and antisense sequences, this hairpin had a double-stranded region of 200 consecutive basepairs corresponding to GUS sequences.
  • the sense and antisense sequences in the expression cassette were each flanked by BamHI and HindHI restrictions sites present at the 5’ and 3’ ends, respectively, relative to the GUS sense sequence.
  • the nucleotides corresponding to these sites were also capable of hybridising, extending the double-stranded region by 6bp at each end.
  • the hairpin RNA structure prior to any processing by Dicer or other RNAses was predicted to have a loop structure of 39 nucleotides.
  • the nucleotide sequence of the hairpin RNA structure including its loop is provided as SEQ ID NO: 15, and its free energy of folding was predicted to be -471.73 kcal/mol. This was therefore an energetically stable hairpin structure.
  • the free energy was calculated using “RNAfold” (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi) based on the nucleotide sequences after the splicing out of the PDK intron sequence.
  • the resultant hairpin RNAs were embedded in a larger RNA molecule with 8 nucleotides added to the 5’ end and approximately 178 nucleotides added at the 3’ end, without considering addition of any poly-A tail at the 3’ end. Since the same promoter-terminator design was used for the modified hairpin RNAs, those molecules also had these extensions at the 5’ and 3’ ends. The length of the hairpin RNA molecules after splicing of the PDH intron was therefore approximately 630 nucleotides.
  • a DNA fragment comprising the modified sequence was then excised by digestion with Xhol and Kpn I and inserted into the XhoVKpnl sites of the base vector pMBW606.
  • This expression cassette was excised with Nol I digestion and inserted into the Notl site of pART27, resulting in the vector designated hpGUS[G:U], encoding the G:U basepaired hairpin RNA molecule.
  • This cassette encoded a hairpin RNA targeting the GUS mRNA and which, when self-annealed by hybridisation of the 200nt sense and antisense sequences, had 52 G:U basepairs (instead of G:C basepairs in hpGUS[wt]) and 148 canonical basepairs, i.e. 26% of the nucleotides of the double- stranded region were involved in G:U basepairs.
  • the 148 canonical basepairs in hpGUS[G:U] were the same as in the control hairpin RNA, in the corresponding positions, including 49 U:A basepairs, 45 A:U basepairs and 54 G:C basepairs.
  • the antisense nucleotide sequence of hpGUS[G:U] was thereby identical in length (200nt) and sequence to the antisense sequence of the control hairpin RNA hpGUS[wt].
  • the hairpin RNA structure prior to any processing by Dicer or other RNAses was predicted to have a loop structure of 45 nucleotides.
  • the nucleotide sequence of the hairpin structure including its loop is provided as SEQ ID NO: 16, and its free energy of folding was predicted to be -331.73 kcal/mol.
  • hpGUS[wt] this was therefore an energetically stable hairpin structure, despite the 52 G:U basepairs which individually are much weaker than the G:C basepairs in hpGUS[wt].
  • the DNA fragment was assembled by annealing the overlapping oligonucleotides GUS-4M-F (SEQ ID NO:56) and GUS-4M-R (SEQ ID NO:57) and PCR extension of 3’ ends using LongAmp Taq polymerase.
  • the amplified DNA fragment was inserted into the pGEM-T Easy vector and the correct nucleotide sequence (SEQ ID NO: 12) was confirmed by sequencing.
  • a DNA fragment comprising the modified sequence was then excised by digestion with Xhol and Kpnl and inserted into the XhoVKpnl sites of the base vector pMBW606.
  • This expression cassette was excised with Notl digestion and inserted into the Notl site of pART27, resulting in the vector designated hpGUS[l:4], encoding the 1:4 mismatched hairpin RNA molecule.
  • This cassette encoded a hairpin RNA targeting the GUS mRNA and which, when self-annealed by hybridisation of the sense and antisense sequences, had mismatches for 50 nucleotides of the 200nt antisense sequence, including the mismatch for the nucleotide at position 200.
  • the double-stranded region of the hairpin RNA had 150 canonical basepairs and 49 mismatched nucleotide pairs over a length of 199nt sense and antisense sequences, i.e. 24.6% of the nucleotides of the double- stranded region were predicted to be mismatched (not involved in basepairs).
  • the hairpin RNA structure prior to any processing by Dicer or other RNAses was predicted to have a loop structure of 45 nucleotides.
  • the nucleotide sequence of the hairpin structure including its loop is provided as SEQ ID NO: 17, and its free energy of folding was predicted to be -214.05 kcal/mol. As for hpGUS[wt], this was therefore an energetically stable hairpin structure, despite the mismatched nucleotides.
  • Each 9th and 10 th nucleotide in each block of 10 nucleotides was substituted by changing C’s to G’s, G’s to C’s, A’s to T’s and T’s to A’s, leaving the other nucleotides unchanged.
  • the DNA fragment was assembled by annealing the overlapping oligonucleotides GUS-10M-F (SEQ ID NO:58) and GUS-10M-R (SEQ ID NO:59) and PCR extension of 3’ ends using LongAmp Taq polymerase.
  • the amplified DNA fragment was inserted into pGEM-T Easy and the correct nucleotide sequence (SEQ ID NO: 13) was confirmed by sequencing.
  • a DNA fragment comprising the modified sequence was then excised by digestion with Xhol and Kpnl and inserted into the XhoVKpnl sites of the base vector pMBW606.
  • This expression cassette was excised with Nol I digestion and inserted into the Notl site of pART27, resulting in the vector designated hpGUS[2:10], encoding the 2:10 mismatched hairpin RNA molecule.
  • This cassette encoded a hairpin RNA targeting the GUS mRNA which, when self-annealed by hybridisation of the sense and antisense sequences, had mismatches for 50 nucleotides of the 200nt antisense sequence, including mismatches for the nucleotides at positions 199 and 200.
  • the double- stranded region of the hairpin RNA had 160 canonical basepairs and 19 di-nucleotide mismatches over a length of 198nt sense and antisense sequences, i.e. 19.2% of the nucleotides of the double-stranded region were predicted to be mismatched (not involved in basepairs).
  • the 160 basepairs in hpGUS[2:10] were the same as in the control hairpin RNA, in the corresponding positions, including 41 U: A basepairs, 34 A:U basepairs, 42 G:C and 43 C:G basepairs.
  • the hairpin RNA structure prior to any processing by Dicer or other RNAses was predicted to have a loop structure of 45 nucleotides.
  • the nucleotide sequence of the hairpin structure including its loop is provided as SEQ ID NO: 18, and its free energy of folding was predicted to be -302.78 kcal/mol.
  • SEQ ID NO: 18 The nucleotide sequence of the hairpin structure including its loop is provided as SEQ ID NO: 18, and its free energy of folding was predicted to be -302.78 kcal/mol.
  • hpGUS[wt] this was therefore an energetically stable hairpin structure, despite the mismatched nucleotides which were expected to bulge out of the stem of the hairpin structure.
  • Plants of the species Nicotiana tabacum (tobacco) transformed with a GUS target gene were used to test the efficacy of the four hairpin RNA constructs described above.
  • the target plants were from two homozygous, independent transgenic lines, PPGH11 and PPGH24, each containing a single-copy insertion of a GUS transgene from a vector pWBPPGH which is shown schematically in Figure 11.
  • the GUS gene in the T-DNA of pWBPPGH had a GUS coding region (nucleotides 7- 1812 of SEQ ID NO:8) operably linked to a 1.3 kb long promoter of the phloem protein 2 (PP2) gene from Cucurbita pepo L.
  • PP2 phloem protein 2
  • the construct pWBPPGH was made by excising the PP2 promoter plus the 5’ UTR and 54 nucleotides of the PP2 protein coding region, encoding the first 18 amino acids of PP2, from the lambda genomic clone CPP1.3 (Wang, 1994), and fusing this fragment with the GUS coding sequence starting with the nucleotides encoding the 3rd amino acid of GUS, generating an N-terminal fusion polypeptide having GUS activity.
  • the pPP2::GUS:Nos-T cassette was inserted into pWBVec2a (Wang et ah, 1998) to generate pWBPPGH, which was used to transform plants of Nicotiana tabacum cv. Wisconsin 38 using Agrobacterium tumefaciens- mediated leaf disk transformation (Ellis et ah, 1987), selecting for resistance to hygromycin.
  • GUS activities in homozygous progeny plants of two transgenic lines PPGH11 and PPGH24 were similar. GUS expression in both transgenic plants was not restricted to phloem but present in most tissues of the plants. GUS expression from the PP2 promoter in these plants therefore appeared to be constitutive.
  • the PP2-GUS plants are two reasons for choosing the PP2-GUS plants as the testing plants: i) they give constitutively high levels of GUS expression about the same as to a 35S-GUS plant; ii) the PP2 promoter is an endogenous PP2 gene promoter derived from Cucurbita pepo with a different sequence to the 35S promoter used to drive the expression of the hpRNA transgenes, which therefore would not be subject to transcriptional cosuppression by the incoming 35S promoter.
  • RNA constructs All four hairpin RNA constructs (Example 6) were used to transform PPGH11 and PPGH24 plants using the Agrobacterium- mediated leaf-disk method (Ellis et ah, 1987), using 50 mg/L kanamycin as the selective agent.
  • Regenerated transgenic plants containing the T- DNAs from the hpGUS constructs were transferred to soil for growth in the greenhouse and maintained for about 4 weeks before assaying for GUS activity.
  • the transgenic plants were healthy and actively growing and in appearance were identical to non-transformed control plants and the parental PPGH11 and PPGH24 plants.
  • 59 transgenic plants were obtained that were transformed with the T-DNA encoding hpGUS[wt]
  • 74 plants were obtained that were transformed with the T-DNA encoding hpGUS [G:U]
  • 33 plants were obtained that were transformed with the T- DNA encoding hpGUS [1:4]
  • 41 plants were obtained that were transformed with the T-DNA encoding hpGUS [2: 10].
  • GUS expression levels were measured using the fluorimetric 4- methylumbelliferyl b-D-glucuronide (MUG) assay (Jefferson et ah, 1987) following the modified kinetic method described in Chen et al. (2005). Plants were assayed by taking leaf samples of about 1cm diameter from three different leaves on each plant, choosing leaves which were well expanded, healthy and green. Care was taken that the test plants were at the same stage of growth and development as the control plants. Each assay used 5 mg protein extracted per leaf sample and measured the rate of cleavage of MUG as described in Chen et al. (2005).
  • MUG fluorimetric 4- methylumbelliferyl b-D-glucuronide
  • the hpGUS[G:U] construct induced consistent and uniform silencing across the independent transgenic lines, with 71 of the 74 plants (95.9%) that were tested showing strong GUS silencing.
  • all of the 33 hpGUS[l:4] plants tested showed reduced levels of GUS activity, with only 8 (24%) yielding ⁇ 10% of the GUS activity relative to the control plants, and the other 25 classified as having weaker silencing.
  • the hpGUS[2:10] construct performed more like the hpGUS[wt] construct, inducing good levels of silencing in some lines (28 of 41, or 68.3%) and gave little or no GUS silencing in the remaining 13 plants.
  • the hpGUS[wt] plants showed the highest average extent of silencing, followed in order by the hpGUS[G:U] plants and the hpGUS[2:10] plants ( Figure 13).
  • the hpGUS[l:4] plants showed the least average reduction in GUS activity.
  • the extent of GUS silencing showed a good correlation with the thermodynamic stability of the predicted hpRNA structures derived from the four different hpRNA constructs (Example 6).
  • Progeny containing the hpGUS [wt] transgenes obviously fell into two categories, namely those that had strong GUS silencing and others that showed weak or no silencing. These classes correlated well with the phenotype of the previous generation, showing that the extent of target gene silencing was heritable. All of the plants in the hpGUS [G:U] lines tested consistently showed strong silencing, whilst the plants in the hpGUS [1:4] lines consistently showed weaker silencing. The inventors concluded that the phenotypes observed in the parental generation were generally maintained in the progeny plants.
  • FIG. 15 An autoradiograph of a hybridised blot is shown in Figure 15. Each lane showed from one to five or six hybridising bands. No two lanes showed the same pattern i.e. the autoradiograph showed that the 16 representative hpGUS[G:U] plants each had different patterns of HindHI fragments that hybridized and therefore came from different transgene insertions.
  • the inventors concluded that the uniform GUS silencing observed for hpGUS [G:U] lines was not due to similar transgene insertion patterns in the plants, and that the uniformity of silencing was caused by the structure of the hpGUS[G:U] RNA. The inventors also concluded that multiple copies of the hpGUS[G:U] transgene were not required in order to obtain strong gene silencing; a single copy of the transgene was sufficient.
  • RNA isolation experiments were carried out on RNA isolated from leaves of the transgenic plants.
  • the Northern blot experiments were carried out to detect the shorter RNAs (sRNA, approx 21-24 nucleotides in length) which resulted from Dicer-processing of the hairpin RNAs.
  • the experiment was carried out on small RNA isolated from transgenic hpGUS[wt] and hpGUS[G:U] plants which also containing the GUS target gene which was expressed as a (sense) mRNA.
  • sRNA short RNA isolated from transgenic hpGUS[wt] and hpGUS[G:U] plants which also containing the GUS target gene which was expressed as a (sense) mRNA.
  • RNA samples were isolated using the hot-phenol method (Wang et al., 2008), and Northern blot hybridization was performed according to Wang et al. (2008), with gel electrophoresis of the RNA samples carried out under denaturing conditions.
  • the probes used were 32P-labelled RNAs corresponding to either the sense sequence or the antisense sequence corresponding to nucleotides 804-1003 of SEQ ID NO:8.
  • FIG. 16 An autoradiograph of a Northern blot, hybridised with either the antisense probe (upper panel) to detect sense sRNA molecules derived from the hairpin RNAs, or hybridised with the sense probe to detect the antisense sRNAs (lower panel), is shown in Figure 16.
  • the Figure shows a qualitative score for the level of GUS expression relative to the control plants lacking the hpGUS constructs. Hybridisation to small RNAs of about 20-25 nucleotides was observed, based on the mobility of the sRNAs compared to RNAs of known length in other experiments.
  • the hpGUS[wt] lines showed a range of variation in the amount of sRNA accumulation.
  • the sense and antisense sRNAs were not as clear as the sense bands. Since the hpGUS [wt] plants contained both the hpGUS transgene, expressing both sense and antisense sequences corresponding to the 200nt target region, and the GUS target gene expressing the full-length sense gene, the sense sRNAs could have been generated from either the hairpin RNA or the target mRNA. There appeared to be negative correlation between the level of sRNA and the degree of GUS silencing in the hpGUS [wt] plants. For example, the two plants represented in lanes 4 and 5 accumulated relatively more sRNA but showed only a moderate extent of GUS downregulation. In contrast, the two plants represented in lanes 7 and 8 had strong GUS silencing but accumulated relatively low levels of sRNA.
  • the hpGUS[G:U] plants accumulated uniform amounts of antisense sRNAs across the lines. Furthermore, the degree of GUS silencing appeared to show good correlation with the amount of antisense sRNA. Almost no sense sRNAs were detected in these plants. This was expected since the RNA probe used in the Northern blot hybridisation was transcribed from the wild-type GUS sequence and therefore had a lower level of complementarity to sense sRNAs from hpGUS[G:U] where all C nucleotides were replaced with U nucleotides, allowing only lower stringency hybridisation. However, this experiment did not exclude the possibility that the hpGUS[G:U] RNA was processed to produce less sense sRNAs or that they were degraded more quickly.
  • hpGUS[G:U] RNA molecule was processed by one or more Dicer enzymes to produce sRNAs, in particular the production of antisense sRNAs which are thought to be mediators of RNA interference in the presence of various proteins such as Argonaute.
  • the observed production of antisense sRNAs implied that the sense sRNAs were also produced, but the experiments did not distinguish between degradation/instability of the sense sRNAs or the lack of detection of sense sRNAs due to insufficient hybridisation with the probe that was used. From these experiments, the inventors also concluded that there were clear differences between the hpGUS[wt] and hpGUS[G:U] RNA molecules in their processing. This indicated that the molecules were recognised differently by one or more Dicers.
  • Example 8 Analysis of sRNAs from transgenic plants expressing modified hairpin RNAs
  • RNA populations from the hpGUS[wt] and hpGUS[G:U] were analysed by deep sequencing of the total, linker-amplifiable sRNAs isolated from the plants.
  • the frequency of sRNAs which mapped to the double- stranded regions of the hairpin RNAs was determined.
  • the length distribution of such sRNAs was also determined.
  • the results showed that there was an increase in the frequency of 22-mer antisense RNAs from the hpGUS[G:U] construct relative to the hpGUS[wt] construct.
  • the increase in the proportion of sRNAs of 22 nt in length indicated a shift in processing of the hpGUS[G:U] hairpin by Dicer-2 relative to hpGUS[wt]
  • McrBC is a commercially available endonuclease which cleaves DNA containing methylcytosine ( m C) bases on one or both strands of double-stranded DNA (Stewart et al., 2000). McrBC recognises sites on the DNA which consist of two half-sites of the form 5’ (G or A) m C 3’, preferably G m C. These half- sites may be separated by several hundred basepairs, but the optimal separation is from 55 to about 100 bp. Double- stranded DNA having such linked G m C dinucleotides on both strands serve as the best substrate.
  • McrBC activity is dependent on either one or both of the GC dinucleotides being methylated. Since plant DNA can be methylated at the C in CG, CHG or CHH sequences where H stands for A, C or T (Zhang et al., 2018), digestion of DNA using McrBC with subsequent PCR amplification of gene-specifc sequences can be used to detect the presence or absence of m C in specific DNA sequences in plant genomes. In this assay, PCR amplification of McrBC-digested genomic DNA which is methylated yields reduced amounts of the amplification product compared to DNA which is not methylated, but will yield an equal amount of PCR product as untreated DNA if the DNA is not methylated.
  • Genomic DNA was isolated by standard methods from plants containing the hpGUS[wt], hpGUS[G:U] or hpGUS[l:4] construct in addition to the target GUS gene (Draper and Scott, 1988). Purified DNA samples were treated with McrBC (Catalog No. M0272; New England Biolabs, Massachusetts) according to the manufacturer’s instructions, including the presence of Mg 2+ ion and GTP required for endonuclease activity. In summary, approximately 1 pg of genomic DNA was digested with McrBC overnight in a 30pl reaction volume. The digested DNA samples were diluted to IOOmI and regions of interest were PCR- amplified as follows.
  • the treated DNA samples were used in PCR reactions using the following primers.
  • 35S-GUS junction sequence for hpGUS [wt] Forward primer (35S- F3), 5 ’ -TGGCTCCTACAAATGCC ATC-3 ’ (SEQ ID NO:60); Reverse primer (GUSwt-R2), 5 ’ -CARRAACTRTTCRCCCTTCAC-3 ’ (SEQ ID NO:61).
  • PCR reactions were performed with the following cycling conditions: 94°C for 1 min, 35 cycles of 94°C for 30 sec, 55°C annealing for 45 sec, 68°C extension for 1 min, and final extension at 68°C for 5 min.
  • PCR amplification products were electrophoresed and the intensity of the bands quantitated.
  • This proximal promoter sequence was important for expression of the transgene and methylation at this region would be likely to reduce expression of the silencing construct through transcriptional gene silencing (TGS) of the transgene. This is termed “self-silencing”.
  • both of the populations of hpGUS[wt] and hpGUS[2:10] transgenic plants showed a wide range in the extent target gene silencing.
  • both of the populations containing hpGUS[G:U] and hpGUS[l:4] plants displayed relatively uniform GUS silencing in many independent lines, with strong silencing observed by the former construct and relatively weaker but still substantial reduction in gene activity by the latter construct.
  • the hairpin RNAs from the [G:U] and [1:4] constructs about 25% of the nucleotides in the sense and antisense sequences were either involved in G:U basepairs or in a sequence mismatch that were evenly distributed across the 200 nucleotide sense/antisense sequences.
  • the mismatches in the DNA constructs between the sense and antisense “arms” or the inverted request structure were considered to significantly disrupt that inverted-repeat DNA structure. Repetitive DNA structures may attract DNA methylation and silencing in various organisms (Hsieh and Fire, 2000).
  • the hpGUS[2:10] construct also comprised mismatches between the sense and antisense region, but each of the 2bp mismatches between the sense and antisense sequences were flanked by 8-bp consecutive matches, so the mismatches may not have disrupted the inverted repeat DNA structure as much as in the [G:U] and [1:4] transgenes.
  • the uniformity of the GUS silencing induced by the hpGUS[G:U] and hpRNA[l:4] might therefore have been due, at least in part, to disruption of the inverted-repeat DNA structure that resulted in less methylation and therefore reduced the self-silencing of the two transgenes.
  • Another benefit of the mismatches between the sense and antisense DNA regions was that cloning of the inverted repeat in E. coli was aided since the bacteria tend to delete or re-arrange perfect inverted repeats.
  • Thermodynamic stability ofhpRNA is important for the degree of target gene silencing
  • the hpGUS[wt] plants had the greatest extent of target gene downregulation, followed in order by hpGUS[G:U], hpGUS[2:10] and hpGUS[l:4].
  • RNAFold analysis predicted that the hpGUS[wt] hairpin RNA structure had the lowest free energy, i.e. the greatest stability, followed by hpGUS[G:U], hpGUS[2:10] and hpGUS[l:4] hairpins.
  • the hpGUS[G:U] RNA was efficiently processed by Dicer
  • sRNAs small RNAs
  • GUS silencing showed relatively poor correlation with the level of sRNA for the hpGUS[wt] construct, with some strongly silenced lines containing relatively low amounts of sRNA. This suggested that GUS silencing in some of the hpGUS[wt] lines was due at least in part to transcriptional silencing rather than sRNA-directed PTGS.
  • the inventors recognised that the self-silencing of the hairpin-encoding gene, which involved methylation of the gene sequences such as the promoter region, was lessened by using the modified hairpin RNA constructs, particularly the G:U construct.
  • McrBC digestion-PCR analysis showed that DNA methylation levels in the 240 bp 35S sequence near the transcription start site (TSS) was reduced in the hpGUS[G:U] and hpGUS[l:4] transgenic populations relative to the hpGUS[wt] population.
  • TSS transcription start site
  • the EIN2 gene (SEQ ID NO: 19) encodes ethylene-insensitive protein 2 (EIN2) which is a central factor in signalling pathways regulated by the plant signalling molecule ethylene, i.e.
  • CHS enzyme chalcone synthase
  • SEQ ID NO:20 encodes the enzyme chalcone synthase (CHS) which is involved in anthocyanin production in the seedcoat in A. thaliana.
  • CHS enzyme chalcone synthase
  • Another G:U modified construct was produced which simultaneously targeted both of the EIN2 and CHS genes, in which the EIN2 and CHS sequences were transcriptionally fused to produce a single hairpin RNA.
  • three additional constructs were made targeting either EIN2, CHS or both EIN2 and CHS, in which cytidine bases in both the sense and antisense sequences were replaced with thymidine bases (herein designated a G:U/U:G construct), rather than in just the sense sequence as done for the modified hairpins targeting GUS.
  • the modified hairpin RNA constructs were tested for their ability to reduce the expression of the endogenous EIN2 gene or the EIN2 and CHS genes using a gene-delivered approach to provide the hairpin RNAs to the cells.
  • the conventional hairpin RNAs used as the controls in the experiment had a double- stranded RNA region of 200 basepairs in length for targeting the EIN2 or CHS mRNAs, singly, or a chimeric double- stranded RNA region comprising 200 basepairs from each of the EIN2 and CHS genes which were fused together as a single hairpin molecule.
  • the EIN2 double- stranded portion was adjacent to the loop of the hairpin and the CHS region was distal to the loop. All of the basepairs in the double- stranded region of the control hairpin RNAs were canonical basepairs.
  • DNA fragments spanning the 200 bp regions of the wild-type EIN2 (SEQ ID NO: 19) and CHS cDNAs (SEQ ID NO:20) were PCR-amplified from Arabidopsis thaliana Col-0 cDNA using the oligonucleotide primer pairs EIN2wt-F (SEQ ID NO:66) and EIN2wt-R (SEQ ID NO:67) or CHSwt-F (SEQ ID NO:68) and CHSwt-R (SEQ ID NO:69), respectively.
  • the fragments were inserted into pGEMT-Easy as for the GUS hairpin constructs (Example 6).
  • DNA fragments comprising the 200 bp modified sense EIN2[G:U] (SEQ ID NO:22) and CHS[G:U] (SEQ ID NO:24) fragments or the 200 bp modified antisense EIN2[G:U] (SEQ ID NO:25) and modified antisense CHS[G:U] (SEQ ID NO:26) fragments, each flanked by restriction enzyme sites, were assembled by annealing of the respective pairs of oligonucleotides, EIN2gu- F + EIN2gu-R, CHSgu-F + CHSgu-R, asEIN2gu-F + asEIN2gu-R, and asCHSgu-F + asCHSgu-R (SEQ ID NOs:70-77), followed by PCR extension of 3’ ends using LongAmp Taq polymerase.
  • the 35S::sense fragment::PDK intron::antisense fragment: :OCS-T cassettes were prepared in an analogous manner as for the hpGUS constructs.
  • the antisense fragments were excised from the respective pGEM-T Easy plasmids by digestion with HindHI and Ba iHI, and inserted into pKannibal between the BamHI and HindHI sites so they would be in the antisense orientation relative to the 35S promoter.
  • the sense fragments were then excised from the respective pGEM-T Easy plasmid using Xhol and Kpnl and inserted into the same sites of the appropriate antisense-containing clone. All of the cassettes in the pGEM-T Easy plasmids were then excised with Not! and inserted into pART27 to form the final binary vectors for plant transformation.
  • the predicted free energy of formation of the hairpin RNAs was estimated by using the FOLD program. These were calculated as (kcal/mol): hpEIN2[wt], -453.5; hpEIN2[G:U], -328.1; hpCHS[wt], -507.7; hpCHS[G:U] -328.5; hpEIN2[G:U/U:G], - 173.5; hpCHS[G:Y/U:G], -186.0; hpCHS::EIN2[wt], -916.4; hpCHS::EIN2[G:U], - 630.9; hpCHS::EIN2[G:U/U:G), -333.8.
  • EIN2 is a gene in A. thaliana that encodes a receptor protein involved in ethylene perception. The gene is expressed in seedlings soon after germination of seeds as well as later in plant growth and development. EIN2 mutant seedlings exhibit hypocotyl elongation relative to isogenic wild-type seedlings when germinated in the dark in the presence of 1-aminocyclopropane-l -carboxylic acid (ACC), an intermediate in the synthesis of ethylene in plants. EIN2 gene expression and the extent of silencing in the transgenic plants was therefore assayed by germinating seed on MS medium containing 50 pg/L of ACC in total darkness and measuring their hypocotyl length, compared to the wild-type seedlings.
  • ACC 1-aminocyclopropane-l -carboxylic acid
  • hypocotyl length was an easy phenotype to measure and was a good indicator of the extent of reduction in EIN2 gene expression, indicating different levels of EIN2 silencing. Plants with silenced EIN2 gene expression were expected to have various degrees of hypocotyl elongation depending on the level of EIN2 silencing, somewhere in the range between wild-type seedlings (short hypocotyls) and null-mutant seedlings (long hypocotyls). Seeds from 20 randomly selected, independently transformed plants for each construct were assayed. Seeds from one plant of the 20 containing the hpCHS::EIN2[G:U] construct did not germinate. The data for hypocotyl length are shown in Figure 27.
  • the hpEIN2[wt] lines showed a considerable range in the extent of EIN2 silencing, with 7 lines (plant lines 2, 5, 9, 10, 12, 14, 16 in Figure 27) clearly showing low levels of silencing or the same hypocotyl length relative to the wild-type, and the other 13 lines having moderate to strong EIN2 silencing.
  • Individual plants within each independent line tended to exhibit a range in the extent of EIN2 silencing, as indicated by differences in hypocotyl length.
  • only two lines (plant lines 5, 18 in Figure 27) comprising the hpEIN2[G:U] construct showed weak EIN2 silencing, with the remaining 18 showing uniform, strong EIN2 silencing.
  • transgenic hpEIN2[wt] and hpEIN2[G:U] populations also differed in the relationship between the extent of EIN2 silencing and the transgene copy number.
  • the transgene copy number was indicated by the segregation ratios for the kanamycin resistance marker gene in progeny plants- a 3:1 ratio of resistant: susceptible seedlings indicating a single locus insertion, whereas a ratio that was much higher indicated multi-loci transgene insertions.
  • the EIN2 gene was also silencing in the seedlings transformed with the CHS::EIN2 fusion hairpin RNA. Similar to the plants containing the single hpEIN2[G:U] construct, the hpCHS::EIN2[G:U] seedlings clearly showed more uniform EIN2 silencing across the independent lines than the hpCHS::EIN2[wt] seedlings. The silencing among individual plants within an independent line also appeared to be more uniform for the hpCHS::EIN2[G:U] lines than the hpCHS::EIN2[wt] lines.
  • Transgenic plants were assayed for the level of CHS gene expression by quantitative reverse transcription PCR (qRT-PCR) on RNA extracted from the whole plants, grown in vitro on tissue culture medium.
  • the primers used for the CHS mRNA were: forward primer (CHS-200-F2), 5 ’ -GAC ATGCCTGGTGCTGACTA-3 ’ (SEQ ID NO:78); reverse primer (CHS-200-R2) 5 ’ -CCTTAGCGATACGGAGGACA-3 ’ (SEQ ID NO:79).
  • the primers used for the reference gene Actin2 used as a standard were: Forward primer (Actin2-For) 5’-TCCCTCAGCACATTCCAGCA-3’ (SEQ ID NO:80) and reverse primer (Actin2-Rev) 5 ’ -GATCCCATTC ATAAAACCCCAG-3 ’ (SEQ ID NO:81).
  • the 35S promoter may not have been sufficiently active in the developing seed coat to provide the level of reduction in CHS activity to provide for the pale seed phenotype seen in null mutants. Improvement in the visual seed coat colour phenotype could be gained by using a promoter that is more active in the seed coat of the seed.
  • Another Arabidopsis gene was selected as an exemplary target gene, namely the phytoene desaturase (PDS) gene which encodes the enzyme phytoene desaturase that catalyzes the desaturation of phytoene to zeta-carotene during carotenoid biosynthesis. Silencing of PDS was expected to result in photo-bleaching of Arabidopsis plants, which could easily be observed visually.
  • a G:U-modified hpRNA construct was therefore made and tested in comparison to a traditional hpRNA constructs targeting a 450 nucleotide PDS mRNA sequence.
  • the 450 nucleotide PDS sequence contained 82 cytosines (C) which were substituted with thymidines (T), resulting in 18.2% of the basepairs in the dsRNA region of the hpRNA hpPDS[G:U] being G:U base pairs.
  • C cytosines
  • T thymidines
  • the genetic construct encoding hpPDS[G:U] and the control genetic construct encoding hpPDS[WT] were introduced into Arabidopsis thaliana Col-0 ecotype using Agrobacterium- mediated transformation.
  • hpPDS[WT] and hpPDS[G:U] constructs 100 and 172 transgenic lines were identified, respectively. Strikingly, all these lines showed photo-bleaching in the cotyledons of young T1 seedlings that emerged on kanamycin-resistant selective medium, with no obvious difference between the two transgenic populations at this early stage of plant growth. These indicated that the two constructs were equally effective at inducing PDS silencing in cotyledons. However, some of the T1 plants developed true leaves that were no longer photo-bleached and looked green or pale green, indicating that PDS silencing was released or weakened in the true leaves.
  • the proportion of transgenic lines showing green true leaves were much higher for the hpPDS[WT] population than for the hpPDS[G:U] population.
  • the transgenic plants were grouped into three different categories based on strong PDS silencing (strong photo-bleaching in whole plant), moderate PDS silencing (pale green or mottled leaves) and weak PDS silencing (fully green or weakly mottled leaves).
  • strong PDS silencing strong photo-bleaching in whole plant
  • moderate PDS silencing pale green or mottled leaves
  • weak PDS silencing fully green or weakly mottled leaves.
  • the proportion of plants with weak PDS silencing was 43% for the hpPDS[WT] lines, compared to 7% for the hpPDS[G:U] lines.
  • the PDS silencing results indicated a developmental variability of hpRNA transgene-induced gene silencing in plants that has not been noted before, and suggested that hpRNA transgene silencing was more efficient and stable in cotyledons than in true leaves.
  • the PDS silencing result suggested that the G:U-modified hpRNA transgene was developmental ⁇ more stable than the conventional hpRNA construct, providing more stable and long-lasting silencing.
  • Northern blot hybridisation was carried out on RNA samples to detect antisense sRNAs from hpEIN2[G:U] plants and to compare their amount and their sizes to sRNAs produced from hpEIN2[wt].
  • the probe was a 32 P-labelled RNA probe corresponding to the 200 nucleotide sense sequence in the hpEIN2[wt] construct and hybridisation was carried out under low stringency conditions to allow for the detection of shorter (20-24 nucleotides) sequences.
  • the autoradiograph from the probed Northern blot is shown in Figure 29.
  • RNA populations from the hpEIN2[wt] and hpEIN2[G:U] are analysed by deep sequencing of the total sRNA populations isolated from whole plants.
  • the proportion of each population that mapped to the double- stranded regions of the hpEIN2[wt] and hpEIN2[G:U] was determined. From about 16 million reads in each population, about 50,000 sRNAs mapped to the hpEIN2[wt] double-stranded region, whereas only about 700 mapped to hpEIN2[G:U]. This indicated that many fewer sRNAs were generated from the [G:U] hairpin. An increase in the proportion of EIN2- specific 22-mers was also observed.
  • Figure 29 showed that both the traditional (fully canonically basepaired) and the G:U-modified hpRNA lines accumulated two dominant size fractions of siRNAs. Consistent with previous reports, the dominant siRNAs from the traditional hpRNA lines migrated similarly to the 21 and 24-nt sRNA size markers. However, the two dominant siRNA bands from both of the G:U modified transgenes migrated slightly faster on the gel, suggesting that they either had a smaller size than, or different terminal chemical modifications to, those from the traditional hpRNA transgenes.
  • RNAs were isolated from one hpGUS[WT] line and two lines each of hpGUS[G:U], hpEIN2[WT] and hpEIN2[G:U] and sequenced using the Illumina platform, resulting in approximately 16 million sRNA reads for each sample. Samples from two strongly silenced hpGUS[l:4] lines were also sequenced. The number of sRNAs which mapped to the double- stranded regions and the intron spacer region of the hairpin RNAs was determined.
  • siRNAs were also mapped to the upstream and downstream regions in the target GUS mRNA and ENI2 mRNA to detect transitive siRNAs.
  • the sequencing data confirmed that hpGUS[G:U] lines, like hpGUS[WT] lines, generated abundant siRNAs, whereas hpGUS[l:4] lines also generated siRNAs but with a much lower abundance.
  • the lower levels of siRNAs from the hpGUS[l:4] lines were consistent with the relatively low efficiency of GUS silencing by hpGUS[l:4] and suggested that the low thermodynamic stability of the dsRNA stem in hpGUS[l:4] RNA reduced Dicer processing efficiency relative to the traditional hairpin.
  • hpEIN2[WT]-7 and hpEIN2[G:U]-14/15 samples showed similar abundance of antisense siRNAs on the Northern blot, but in the sequencing data the hpEIN2[G:U] lines gave much smaller numbers of total 20-24 nt antisense siRNA reads (17,290 and 29,211) than the hpEIN2[WT]-7 line (134,112 reads).
  • the sRNA sequencing data indicated that siRNAs from the traditional and mismatched hpRNA lines had a similar size profile, with the exception of the 22-nt size class, suggesting that the differential migration detected by Northern blot was due to different 5’ or 3’ chemical modifications.
  • the discrepancy in relative sRNA abundance eg. the hpEIN2[WT] vs. hpEIN2[G:U]-derived siRNAs and the 21- nt vs. 24-nt
  • the sequencing data implied that the different siRNA populations and size classes may have different cloning efficiencies during sRNA library preparation.
  • Plant sRNAs are known to have a 2’-0-methyl group at the 3’ terminal nucleotide that is thought to stabilize the sRNAs. This 3’ methylation was previously shown to inhibit, but not prevent, 3’ adaptor ligation reducing sRNA cloning efficiency (Ebhardt et al 2005). Therefore, hpRNA[WT] and hpRNA[G:U] -derived siRNAs were with sodium periodate in b-elimination assays.
  • the standard sRNA sequencing protocol is based on sRNAs having 5’ monophosphate allowing 5’ adaptor ligation (Lau et ah, 2001). Dicer-processed sRNAs were assumed to have 5’ monophosphate but in C. elegans many siRNAs are found to possess di- or tri-phosphate at the 5’ terminus which changes gel mobility of sRNAs and prevents sRNA 5’ adaptor ligation in the standard sRNA cloning procedure (Pak and Fire 2007). Whether plant sRNAs also have differential 5’ phosphorylation was unknown.
  • the 5’ phosphorylation status of the hpRNA[WT] and hpRNA[G:U] -derived siRNAs was therefore examined by treating the total RNA with alkaline phosphatase followed by Northern blot hybridization. This treatment reduced the gel mobility for all hpRNA-derived sRNAs, indicating the presence of 5’ phosphorylation.
  • the hpRNA[G:U] -derived siRNAs showed greater mobility shift than the hpRNA[WT]- derived siRNAs after phosphatase treatment, resulting in the two groups of dephosphorylated siRNAs migrating at the same position on the gel.
  • the 21 and 24-nt sRNA size markers were radio-actively labelled at the 5’ end with 32 P using polynucleotide kinase reaction, and so should have a monophosphorylated 5’ terminus.
  • the siRNAs produced from the traditional and G:U-modifed hpRNA transgenes in plant cells were phosphorylated differently.
  • the primers used for the 35S promoter region Forward primer (Top-35S-F2), 5’- AGAAAATYTTY GTY AAY ATGGTGG-3 ’ (SEQ ID NO:82), reverse primer (Top- 35S-R2), 5 ’ -TCARTRRARATRTC ACATC AATCC-3 ’ (SEQ ID NO:83).
  • Quantitation of the extent of DNA methylation was determined by carrying out Real-Time PCR assays. For each plant, the quotient was calculated: rate of amplification of the DNA fragment after treatment of the genomic DNA with McrBC/ rate of amplification of the DNA fragment without treatment of the genomic DNA with McrBC.
  • the hpEIN2[G:U] lines showed less DNA methylation at both the 35S promoter and the 35S-EIN2 junction. Indeed, four of these 12 G:U lines, corresponding to lanes 1, 2, 3 and 7 in Figure 30 (lanes 13, 14, 15 and 20 in Figure 31), had no obvious DNA methylation as indicated by the equal strength of PCR bands between McrBC-treated and untreated samples. When these amplifications were quantitated by qPCR, six of the 12 lines showed little to no reduction in the fragment from the McrBC treatment and therefore little to no DNA methylation - see lower panel of Figure 31, lines 13, 14, 15, 18, 19 and 20.
  • Double-stranded RNA having G:U basepairs induce more uniform gene silencing than conventional dsRNA
  • both hpEIN2[G:U] and hpCHS:EIN2[G:U] induced more consistent and uniform EIN2 silencing than the respective hpRNA[wt] constructs encoding a conventional hairpin RNA.
  • the uniformity not only occurred across many independent transgenic lines, but also across sibling plants within a transgenic line each having the same transgenic insertion.
  • the extent of EIN2 silencing induced by hpEIN2[G:U] was close to that of strongly silenced hpEIN2[wt] lines.
  • cytosine bases in the CHS sequence occurred in sets of two or three consecutive cytosines, so not all of those need be substituted.
  • another set of CHS constructs are made using a sequence containing a range of cytosine substitutions, from about 5%, 10%, 15%, 20% or 25% cytosine bases substituted. These constructs are tested and an optimal level determined.
  • the hpEIN2[G:U] lines express more uniform levels ofsiRNAs
  • the hpEIN2[G:U] lines accumulated sRNAs with a more uniform level across the independent lines. This confirmed the conclusion with the hpGUS constructs that [G:U] modified hpRNA was efficiently processed by Dicer and capable of inducing effective target gene silencing.
  • Fusion constructs also provide for gene silencing
  • the purpose of including the CHS:EIN2 fusion constructs in the experiment was to test if two target genes could be silenced with a single hairpin-encoding construct.
  • the GUS experiment suggested that the free energy and therefore stability of the hairpin RNA correlated positively with the extent of target gene silencing.
  • the results showed that the CHS:EIN2 fusion construct did result in silencing of both genes - for CHS at least at the mRNA level.
  • hpEIN2[G:U/U:G] and hpCHS:EIN2[G:U/U:G] in which both the sense and antisense sequences were modified from C to T so that 46% of basepairs were converted from canonical basepairs to G:U basepairs, induced only weak or no EIN2 silencing in most of the transgenic plants.
  • Possible explanations include i) there were too many G:U basepairs which resulted in inefficient Dicer processing, and ii) sRNAs binding to target mRNA including too many G:U basepairs did not induce efficient mRNA cleavage, or a combination of factors.
  • hpEIN2[G:U] lines showed little to no promoter methylation and most of the plants analysed showed less methylated cytosines.
  • hpGUS lines several factors may contribute to the reduced methylation: i) the inverted-repeat DNA structure was disrupted by changing C bases to T bases in the sense sequence, and ii) the sense EIN2 sequence lacked cytosines so could not be methylated by sRNA- directed DNA methylation, and iii) a reduced level of production of 24-mer RNAs due to the change in the structure of the dsRNA region with the G:U basepairs, resulting in changes in the recognition by some Dicers and so a decrease in Dicer 3 and/or Dicer 4 activity and relatively more Dicer 2 activity.
  • the hpEIN2[G:U] transgene may behave like a normal, non-RNAi transgene (such as an over-expression transgene) and the promoter methylation observed in some of the lines was due to T-DNA insertion patterns rather than the inherent inverted-repeat DNA structure of a hpRNA transgene.
  • Example 14 Modified hairpins for reducing expression of another endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous endogenous end
  • the FANCM gene in A. thaliana and in Brassica napus encodes a Fanconi Anemia Complementation Group M (FANCM) protein, which is a DEAD/DEAH box RNA helicase protein, Accession Nos and NM_001333162 and XM_018659358.
  • FANCM Fanconi Anemia Complementation Group M
  • the nucleotide sequence of the protein coding region of the cDNA corresponding to the FANCM gene of A. thaliana is provided in SEQ ID NO:31, and for B. napus in SEQ ID NO:32.
  • a target region in the A. thaliana gene was selected: nucleotides 675-1174 (500 nucleotides) of SEQ ID NO:31.
  • a target region in the B. napus gene was selected: nucleotides 896-1395 (500 bp) of SEQ ID NO:32.
  • Nucleotide sequences of the hpFANCM- At[wt] , hpFANCM-At[G:U] , hpFANCM-Bn[wt] and hpFANCM-Bn[G:U] constructs are provided in SEQ ID NOs:33-36.
  • G:U constructs all cytosine bases in the sense sequences were replaced with thymine bases - 102/500 (providing 20.4% G:U basepairs) in the A. thaliana construct and 109/500 (21.8% G:U basepairs) in the B. napus construct.
  • the longest stretch of contiguous canonical basepairing in the double-stranded region of the B. napus G:U modified hairpin was 17 basepairs, and the second longest 16 contiguous basepairs.
  • the DDM1 gene in B. napus encodes a methyltransferase which methylates cytosine bases in DNA (Zhang et al., 2018).
  • Nucleotide sequences of the hpDDMl-Bn[wt] and hpDDMl-Bn[G:U] constructs are provided in SEQ ID NOs:38-39.
  • cytosines in the sense sequences were replaced with thymines - 106/502 (21.1% G:U basepairs) in the B. napus construct.
  • the longest stretch of contiguous canonical basepairing in the double- stranded region of the G:U modified hairpin was 20 basepairs, and the second longest 15 contiguous basepairs.

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